Prevention, treatment and diagnosis of p.gingivalis infection

ABSTRACT

The invention relates to generation and use of cellular and humoral responses for the prevention and treatment of  P. gingivalis  related conditions and diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.17/098,021, filed Nov. 13, 2020, which is a continuation of U.S.application Ser. No. 16/134,721, filed Sep. 18, 2018, which is acontinuation of U.S. application Ser. No. 15/370,144, filed Dec. 6,2016, which is a continuation of U.S. application Ser. No. 14/487,461,filed Sep. 16, 2014, which is a divisional of U.S. application Ser. No.13/060,653, filed Feb. 24, 2011, which is the U.S. National Stage ofInternational Application No. PCT/AU2009/01112, filed Aug. 28, 2009, andwhich claims priority to U.S. Provisional Application No. 61/151,132,filed Feb. 9, 2009, Australian Application No.

2009903052, filed Jun. 30, 2009, Australian Application No. 2008905483,filed Oct. 23, 2008, and Australian Patent Application No. 2008904476,filed Aug. 29, 2008.

SEQUENCE LISTING

The instant application contains a Sequence Listing which is being filedelectronically in XML format and is hereby incorporated by reference inits entirety. Said XML copy, created on Aug. 7, 2023, is namedCorrected_SL_53241969 SCH.xml and is 253,080 bytes in size.

FIELD OF THE INVENTION

The invention relates to peptides and chimeric or fusion proteins and tothe use of these proteins to elicit cellular and humoral responses forthe prevention and treatment of P. gingivalis—related conditions anddiseases.

BACKGROUND OF THE INVENTION

Chronic periodontitis is an inflammatory disease of the supportingtissues of the teeth leading to resorption of alveolar bone and eventualtooth loss. The disease is a major public health problem in allsocieties and is estimated to affect up to 15% of the adult populationwith severe forms affecting 5-6%.

The development and progression of chronic periodontitis has beenassociated with specific Gram-negative bacteria in subgingival plaque.The presence of Porphyromonas gingivalis in subgingival plaque has beenstrongly associated with disease.

The persistence of P. gingivalis in subgingival plaque fromperiodontitis patients after treatment (scaling and root planing) hasbeen reported to be significantly associated with progressive alveolarbone loss. Furthermore an increase in P. gingivalis cell numbers insubgingival plaque has been shown to correlate with disease severity asmeasured by attachment loss, periodontal pocket depth and bleeding onprobing.

Oral infection with P. gingivalis has been shown to induce periodontalbone loss in mice, rats and non-human primates. In addition, there hasbeen increasing linkage of periodontal disease, and of P. gingivalisinfection, with cardiovascular diseases and certain cancers.

A number of virulence factors have been reported to contribute to thepathogenicity of P. gingivalis including; LPS, fimbriae, hemagglutinin,hemolysin and extracellular hydrolytic enzymes (especially the Arg-X andLys-X specific proteinases), otherwise known as “P. gingivalistrypsin-like enzymes”.

The magnitude of the public health problem is such that there is a needfor an antiserum, particularly specific antibodies that provide a strongprotective response to P. gingivalis infection and means for providingsame.

One problem has been that it is not clear how to obtain a strongprotective response to P. gingivalis infection where there are aplethora of virulence factors to select from.

The relative immunogenicity of epitopes amongst virulence factors is notwell understood, nor is the relative immunogenicity of epitopes on agiven factor, particularly where it is not clear as to whether furtherepitopes remain to be identified.

One particular problem has been that many virulence factors are formedfrom multiple domains and are difficult to express so as to present aconformation approaching that found on P. gingivalis. Further, whenthese domains are expressed as discrete units i.e. in isolation of othervirulence factor domains, they tend to fold into a conformationdistinguished from that found on P. gingivalis.

Further, of the many different options for modifying the immunogenicityof a virulence factor it is not clear which would be most likely toprovide for a protective immune response.

In work leading to the present invention the inventors have identifiedpeptides having an amino acid sequence that is the same as, or thatshares homology with, an amino acid sequence that forms a region of a P.gingivalis trypsin—like enzyme, said region defining a site in saidenzyme for cleavage of a peptide bond located C-terminal to Lys or Argin a peptide containing Lys or Arg, and incorporated such a peptide intoa chimeric or fusion protein which, when used as a vaccine, providesbetter protection against periodontal tissue destruction than purifiedproteinase-adhesin complex formed from native P. gingivalis trypsin-likeenzyme or killed whole cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a chimeric or fusionprotein for inducing an immune response to P. gingivalis, the proteinincluding a first peptide joined directly or through a linker to asecond peptide, wherein:

-   -   (A) said first peptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:1; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:2; and    -   (B) said second peptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Lys-X-proteinase of P. gingivalis; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Arg-X-proteinase of P. gingivalis; or        -   (iii) part of, or all of a sequence that is the same as, or            homologous to the sequence of a HagA adhesin domain of P.            gingivalis.

In another aspect, the invention provides a chimeric or fusion proteinfor inducing an immune response to P. gingivalis, the protein includinga peptide joined directly or through a linker to a polypeptide, wherein:

-   -   (A) said peptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:1; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:2; and    -   (B) said polypeptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Lys-X-proteinase of P. gingivalis; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Arg-X-proteinase of P. gingivalis; or        -   (iii) part of, or all of a sequence that is the same as, or            homologous to the sequence of a HagA adhesin domain of P.            gingivalis.

In another aspect, the invention provides a peptide for inducing animmune response to P. gingivalis the peptide having a sequence:

-   -   (i) that is the same as, or homologous to the sequence shown in        one of SEQ ID No: 64 to 66; and    -   (ii) that is the same as, or homologous to the sequence shown in        SEQ ID No: 67 or 68.

In one aspect, the peptide having a sequence that is the same as orhomologous to sequence shown in one of SEQ ID No: 64 to 68 may beprovided in the form of a chimeric or fusion protein in which thepeptide is joined directly or through a linker to a second peptide,wherein the second peptide includes:

-   -   (i) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Lys-X-proteinase of P. gingivalis; or    -   (ii) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Arg-X-proteinase of P. gingivalis; or    -   (iii) part of, or all of a sequence that is the same as, or        homologous to the sequence of a HagA adhesin domain of P.        gingivalis.

In yet another aspect, the invention provides a composition such as anantigenic composition, particularly a vaccine composition, including achimeric or fusion protein or peptide as broadly described above,optionally in association with an adjuvant.

In this aspect, the invention also provides a method of preventing orreducing the incidence or severity of a P. gingivalis—related conditionor disease in a subject, which comprises administering to the subject achimeric or fusion protein as described above, or a composition asdescribed above.

In this aspect, the invention further provides the use of a chimeric orfusion protein as described above, or a composition as described above,in, or in the manufacture of a medicament for preventing or reducing theincidence or severity of a P. gingivalis—related condition or disease ina subject.

In another aspect, the invention provides an antibody, particularly amonoclonal antibody, raised against a chimeric or fusion protein orpeptide as broadly described above.

In this aspect, the invention also provides a method of preventing orreducing the severity of a P. gingivalis-related disease or condition ina subject, which comprises administering to the subject an antibody asdescribed above.

In this aspect, the invention further provides the use of an antibody asdescribed above in, or in the manufacture of a medicament for preventingor reducing the incidence or severity of a P. gingivalis—relatedcondition or disease in a subject.

In yet another aspect, the invention also provides a nucleic acidmolecule including a nucleotide sequence encoding a chimeric or fusionprotein as broadly described above, optionally operatively linked to atleast one regulatory element.

In this aspect, the invention further provides a vector including such anucleic acid molecule, as well as a prokaryotic or eukaryotic cellincluding such a nucleic acid molecule.

In this aspect, the invention also provides a method of preventing orreducing the incidence or severity of a P. gingivalis-related conditionor disease in a subject, which comprises administering to the subject anucleic acid molecule as described above, a vector as described above,or a prokaryotic or eukaryotic cell as described above.

In this aspect, the invention further provides the use of a nucleic acidmolecule as described above, a vector as described above, or aprokaryotic or eukaryotic cell as described above, in, or in themanufacture of a medicament for preventing or reducing the severity of aP. gingivalis-related disease or condition in a subject.

In a further aspect, the invention provides a method for the diagnosisor monitoring of a P. gingivalis-related condition or disease in asubject, which comprises use of a chimeric or fusion protein asdescribed above to detect anti-P. gingivalis antibodies in a biologicalsample from said subject.

In this aspect, the invention also provides the use of a chimeric orfusion protein as described above, to detect anti-P. gingivalisantibodies in a biological sample from a subject.

In yet another aspect, the invention provides a method for the diagnosisor monitoring of a P. gingivalis-related condition or disease in asubject, which comprises use of an antibody as described above, todetect the presence of P. gingivalis in a biological sample from saidsubject.

In this aspect, the invention also provides the use of an antibody asdescribed above, to detect the presence of P. gingivalis in a biologicalsample from a subject.

In another aspect, the invention provides a use of a peptide having partof, or all of a sequence that is the same as, or homologous to asequence of a P. gingivalis Lys-X or Arg-X proteinase, or a nucleic acidencoding said peptide for the manufacture of a chimeric or fusionprotein for inducing an immune response to P. gingivalis. In this aspectthe peptide may have a sequence shown in one of SEQ ID No: 17, 18, 25 or26.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Coomassie blue stain of the SDS-PAGE gel of recombinantKgp Proteins. Lane 1=KAS2-KLA1, Lane 2=KLA1, Lane 3=KsA1, Lane4=KAS1-KsA1. Molecular weight markers are indicated as kDa.

FIG. 2 shows antibody recognition of KAS2 peptide and formalin killed P.gingivalis W50 cells. (A) KAS2 peptide was probed with antisera raisedto formalin killed P. gingivalis W50 cells (FK-W50), recombinantproteins KAS1-KsA1, KAS2-KLA1, and synthetic KAS2-DT conjugate and PBSin an ELISA. (B) formalin killed P. gingivalis W50 cells were probedwith antisera raised to formalin killed P. gingivalis W50 cells(FK-W50), recombinant proteins KAS1-KsA1, KAS2-KLA1, KLA1 and PBS in anELISA. Antibody responses are expressed as the ELISA titre OD415obtained minus double the background level, with each titre representingthe mean±standard deviation of three values.

FIG. 3 shows P. gingivalis-induced horizontal bone loss of maxillaemolars of mice immunised with the recombinant proteins and recombinantchimera proteins, formalin-killed P. gingivalis and adjuvant alone (PBS,IFA) or non-orally infected (non-challenged) mice. In this figureKAS2-KLA1 is shown as AS2-LA1, KLA1 is shown as LA1, KAS1-KsA1 is shownas AS1-sA1, KsA1 is shown as sA1. Measurement of bone loss is the meanof the area measured in millimeters squared (mm2) from the cementoenameljunction (CEJ) to the alveolar bone crest (ABC) of the buccal side ofeach maxillary molar of both the left and right maxillae. Data wasnormally distributed as measured by Levene's homogeneity of variance andare presented as mean (n=12) in mm2 and were analyzed using the One-Wayanalysis of variance and Dunnett's T3 test. *, indicates group hassignificantly (P<0.001) less bone loss than control (infected) group. †,indicates group has significantly (P<0.001) more bone loss than theAS2-LA1 group.

FIG. 4 shows serum antibody subclass responses of immunised mice in theperiodontitis model. Sera from mice; A (pre-oral inoculation) and B(post-oral inoculation) immunised with recombinant proteins KsA1, KLA1,KAS1-KsA1 and KAS2-KLA1 and formalin killed P. gingivalis strain W50were used in the ELISA with the formalin killed P. gingivalis strain W50as the adsorbed antigen. Antibody responses IgG (black bars), IgG1 (greybars), IgG2a (white bars), IgG2b (horizontal striped bars), IgG3(diagonal striped bars), are expressed as the ELISA titre (log 2)obtained minus the background level, with each titre representing themean±standard deviation of three values.

FIGS. 5A-5B show a PEPSCAN analysis of peptide-specific antibodyreactivity to overlapping peptides representing the KAS2 peptidesequence 433-468. (FIG. 5A) KAS2 overlapping peptides (offset 1, overlap7) probed with KAS1-KsA1 (white bars), KAS2-KLA1 (black bars) antisera.(FIG. 5B) KAS2 overlapping peptides (offset, overlap 7) probed withKAS2-DT conjugate antisera. Each bar displays the antibody reactivity(optical density [OD] at 415 nm). FIGS. 5A and 5B each disclose SEQ IDNOS 87, 88, 5, 7, 89-96, 19, 21, 23, 25 and 97-109, respectively, inorder of appearance.

FIG. 6 . Chimera AS2-LA1 induces an antibody response in outbred micethat recognises P. gingivalis whole cells and the RgpA-Kgp complex. CD1outbred mice were immunised with chimera AS2-LA1 (50 mg/mouse) and thecollected sera used in ELISA with AS2-LA1 (A), formalin killed P.gingivalis strain W50 (B) and RgpA-Kgp complex (C) as the absorbedantigens. In this figure KAS2-KLA1 is shown as AS2-LA1.

The titre for each immunoglogulin isotype to each antigen was determinedand the data expressed as the ELISA titre (′000) obtained minus doublethe background level, with each titre representing the mean±standarddeviation of three values.

FIG. 7 . Protein model of the Kgp proteinase. KAS2 [Asn433-Lys468]. (A)KAS4 [Asp388-Va1395] (B), KAS5 [Asn510-Asp516] (C) and KAS6[Ile570-Tyr580] (D).

DETAILED DESCRIPTION OF THE EMBODIMENTS

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

The inventors have found that the regions of P. gingivalis trypsin-likeenzymes that flank or otherwise define a catalytic or active site forcleavage of a peptide bond are highly immunogenic and indeed sufficientto provide for a humoral response to P. gingivalis infection. Inparticular, it has been found that a chimeric or fusion proteinincluding one or more of these regions provides protection againstalveolar bone loss which is greater than that seen for antisera raisedagainst whole cells and other immunogens. The finding is particularlysurprising as, to date, the catalytic domain of trypsin-like enzymes ofP. gingivalis has been found to be relatively weakly immunogenic.

In one aspect, the present invention provides a chimeric or fusionprotein for inducing an immune response to P. gingivalis, the proteinincluding a first peptide joined directly or through a linker to asecond peptide, wherein:

-   -   (A) said first peptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:1; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:2; and        -   (B) said second peptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Lys-X-proteinase of P. gingivalis; or    -   (ii) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Arg-X-proteinase of P. gingivalis; or        -   (iii) part of, or all of a sequence that is the same as, or            homologous to the sequence of a HagA adhesin domain of P.            gingivalis.

As used herein, the term “peptide” is used to refer to an amino acidsequence of up to about 40 amino acid residues, preferably from 5 to 40amino acid residues.

In one embodiment, a polypeptide is used in place of or in other wordsinstead of the “second peptide”. The term “polypeptide” is used to referto an amino acid sequence of at least about 40 amino acid residues.

Thus, in another aspect there is provided a chimeric or fusion proteinfor inducing an immune response to P. gingivalis, the protein includinga peptide joined directly or through a linker to a polypeptide, wherein:

-   -   (A) said peptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:1; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence shown in SEQ ID No:2; and    -   (B) said polypeptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Lys-X-proteinase of P. gingivalis; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Arg-X-proteinase of P. gingivalis; or        -   (iii) part of, or all of a sequence that is the same as, or            homologous to the sequence of a HagA adhesin domain of P.            gingivalis.

In another aspect, the invention provides a peptide for inducing animmune response to P. gingivalis selected from the group consisting of:

-   -   (i) a sequence that is the same as or homologous to the sequence        shown in one of SEQ ID No: 64 to 66; and    -   (ii) a sequence that is the same as or homologous to the        sequence shown in SEQ ID No: 67 or 68.

In an aspect of the invention, where the peptide has a sequence of SEQID No: 64 to 68, the peptide may be provided in the form of a chimericor fusion protein in which the peptide is joined directly or through alinker to a second peptide. In an embodiment, the second peptide of thechimeric or fusion protein includes:

-   -   (i) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Lys-X-proteinase of P. gingivalis; or    -   (ii) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Arg-X-proteinase of P. gingivalis; or    -   (iii) part of, or all of a sequence that is the same as, or        homologous to the sequence of a HagA adhesin domain of P.        gingivalis.

In the above described embodiment a polypeptide is used in place of, orin other words instead of the second peptide. Thus, in another aspectthere is provided a chimeric or fusion protein for inducing an immuneresponse to P. gingivalis, the protein including a peptide joineddirectly or through a linker to a polypeptide, wherein:

-   -   (A) said peptide includes:        -   (i) a sequence that is the same as or homologous to the            sequence shown in one of SEQ ID No: 64 to 66; or        -   (ii) a sequence that is the same as or homologous to the            sequence shown in SEQ ID No: 67 or 68; and    -   (B) said polypeptide includes:        -   (i) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Lys-X-proteinase of P. gingivalis; or        -   (ii) part of, or all of a sequence that is the same as, or            homologous to the sequence of an adhesin domain of the            Arg-X-proteinase of P. gingivalis; or        -   (iii) part of, or all of a sequence that is the same as, or            homologous to the sequence of a HagA adhesin domain of P.            gingivalis.

As used herein, a reference to a “homologue” of a peptide or polypeptideis a reference to a peptide or polypeptide having an amino acid sequencethat shares homology or that is homlogous to, or that has identity withthe amino acid sequence of the first-mentioned peptide or polypeptide,preferably at least 90% sequence identity, more preferably at least 95%and even more preferably at least 98% sequence identity when thecomparison is performed by a BLAST algorithm wherein the parameters ofthe algorithm are selected to give the largest match between therespective sequences over the entire length of the respective referencesequences. Sequence identity refers to exact matches between the aminoacids of two sequences which are being compared. Such a homologue mayderive from a naturally occurring variant or isolate of theLys-X-proteinase or Arg-X-proteinase of P. gingivalis. Alternatively, itmay be a “conservative-substitution” variant of a peptide or polypeptidefrom the Lys-X-proteinase or Arg-X-proteinase of P. gingivalis in whichone or more amino acid residues have been changed without altering theoverall conformation and function of the peptide or polypeptide;including, but by no means limited to, replacement of an amino acid withone having similar properties. Amino acids with similar properties arewell known in the art. For example, polar/hydrophilic amino acids whichmay be interchangeable include asparagine, glutamine, serine, cysteine,threonine, lysine, arginine, histidine, aspartic acid and glutamic acid;nonpolar/hydrophobic amino acids which may be interchangeable includeglycine, alanine, valine, leucine, isoleucine, proline, tyrosine,phenylalanine, tryptophan and methionine; acidic amino acids which maybe interchangeable include aspartic acid and glutamic acid and basicamino acids which may be interchangeable include histidine, lysine andarginine. Preferably such conservative-substitution variants have lessthan 20, more preferably less than 15, more preferably less than 10, andmost preferably less than 5 amino acid changes.

A region of a P. gingivalis trypsin-like enzyme—especially aLys-X-proteinase (Kgp) or Arg-X-proteinase (RgpA)—that defines a site inan enzyme for cleavage of a peptide bond can be determined following theteaching of the specification herein, particularly in relation to FIG. 7and Example 9, which exemplify the process for predictingthree-dimensional conformation of the catalytic site as it appears on P.gingivalis for Lys-X-proteinase. Example 10 provides methodology formodelling of the Arg-X-proteinase three-dimensional conformation.

In certain embodiments, the chimeric or fusion protein, or first orsecond peptide components thereof may be formed from a peptidomimetic. Apeptidomimetic is a molecule that mimics one or more characteristics ofa given peptide, for example conformation, and that consists of aminoacid residues, some of which may not be naturally occurring.

Having identified the immunogenic regions of the catalytic site, theinventors have determined the sequence of various peptide immunogensagainst which a humoral response can be raised. In particular, ‘six’regions that flank or otherwise define the catalytic site have beendefined as follows: KAS1/RAS1, KAS2/RAS2, KAS3/RAS3, KAS4/RAS4,KAS5/RAS5 and KAS6 (see Table 1). With this information, the inventorshave been able to interrogate protein sequence databases to determinepeptides that share homology with amino acid sequences that form regionsthat flank a catalytic site and hence that represent immunogenicepitopes found on P. gingivalis. The sequence of these peptides areidentified by the following structural formula:

TABLE 1 Sequences that flank the active site of Kgp and RgpA.Kgp Lys - X RgpA Arg -X (numbering (numbering according to according toRgpA Arg -X Region SEQ ID No. 62) Kgp Lys - X Consensus SEQ ID No. 61)Consensus PAS1K/ PAS1K (432-453) LNTGVSFANYTAHGS PAS1R (426-446)FNGGISLANYTGHGSET PAS1R ETAWADP AWGT (SEQ ID NO: 34) (SEQ ID NO: 30)KAS1/ KAS1 (432-454) LNTGV[G/S]FANYTAH RAS1 (426-448) FNGGISL[V/A]NYTGHGRAS1 GSET[S/A]WADP[S/L] SETAWGTSH (SEQ ID NO: 27) (SEQ ID NO: 31) KAS2/KAS2 (433-468) NTGV[G/S]FANYTAHG RAS2 (427-462) NGGISL[V/A]NYTGHGS RAS2SET[S/A]WADP[S/L][L/ ETAWGTSHFGTTHVKQ V]T[A/T][T/S]Q[V/L]KAL LTNSNQTNK[D/N]K (SEQ ID NO: 32) (SEQ ID NO: 28) KAS3/ KAS3 (436-455)V[G/S]FANYTAHGSET RAS3 (430-449) ISL[V/A]NYTGHGSETA RAS3[S/A]WADP[S/L][L/V] WGTSHF (SEQ ID NO: 29) (SEQ ID NO: 33) KAS4/KAS4 (388-395) D[S/Y][Y/S]WN[P/S][K/ RAS4 (379-386) EGGPSADN RAS4Q][I/V] (SEQ ID NO: 64) (SEQ ID NO: 67) KAS5/ KAS5 (510-516) NSYWGEDRAS5 (508-514) [N/D]Q[S/Y]WA[S/P]P RAS5 (SEQ ID NO: 65) (SEQ ID NO: 68)KAS6 KAS6 (570-580) IGN[V/I]THIGAHY (SEQ ID NO: 66)

The inventors have found that chimeric proteins including these peptideshave a number of utilities. For example, as described herein, someproduce a humoral response that is highly protective for treatment orprevention of bone loss as observed in chronic periodonitis. Thepeptides may also be used in a diagnostic assay wherein they can detector monitor specificities in an individual's serum, thereby indicatingwhether or not the individual is infected and if so, whether treatmentsare required or if provided, whether they have been effective.

It will be understood that the region of a P. gingivalis trypsin-likeenzyme that defines a site in the enzyme for cleavage of a peptide bondlocated C-terminal to Lys or Arg, does not comprise a complete sequenceof the Lys-X-proteinase or Arg-X-proteinase.

As used herein, the terms “heterologous protein” or “chimeric or fusionprotein” are used to refer to a protein that is composed of functionalunits, domains, sequences or regions of amino acids derived fromdifferent sources or that are derived from the same source and that havebeen assembled so as to have an organisation that is distinguished fromthat observed in a molecule from which the unit, domain, sequence orregion is derived or related to. A common feature of the chimeric orfusion proteins of the invention is that they contain at least onepeptide having an amino acid sequence that is the same as or that shareshomology with a sequence of a P. gingivalis trypsin-like enzyme thatdefines a catalytic site for cleavage of a peptide bond.

In a preferred embodiment, where the first peptide comprises a peptidefrom the Kgp[432-468] region, it is preferably (i) a peptide whichcomprises a sequence selected from VSFANYT (SEQ ID NO: 3) and VGFANYT(SEQ ID NO: 4), more preferably a sequence selected from GVSFANYT (SEQID NO: 5), GVGFANYT (SEQ ID NO: 6), VSFANYTA (SEQ ID NO: 7) and VGFANYTA(SEQ ID NO: 8); or (ii) a peptide which comprises a sequence selectedfrom ETAWAD (SEQ ID NO: 9), ETSWAD (SEQ ID NO: 10), TAWADP (SEQ ID NO:11) and TSWADP (SEQ ID NO: 12), preferably a sequence selected fromSETAWAD (SEQ ID NO: 13), SETSWAD (SEQ ID NO: 14), ETAWADP (SEQ ID NO:15), ETSWADP (SEQ ID NO: 16), TAWADPL (SEQ ID NO: 17) and TSWADPL (SEQID NO: 18), more preferably a sequence selected from GSETAWAD (SEQ IDNO: 19), GSETSWAD (SEQ ID NO: 20), SETAWADP (SEQ ID NO: 21), SETSWADP(SEQ ID NO: 22), ETAWADPL (SEQ ID NO: 23), ETSWADPL (SEQ ID NO: 24),TAWADPLL (SEQ ID NO: 25) and TSWADPLL (SEQ ID NO: 26). More preferably,this peptide is selected from the KAS1[432-454], KAS2[433-468] andKAS3[436-455] peptides shown in Table 1. Alternatively, the firstpeptide may be the PAS1K[432-453] peptide, also known as PAS1(K48),disclosed in International Patent Application No. PCT/AU98/00311 (WO98/049192). The sequence identifiers corresponding to these peptides areshown in Table 3.

Similarly, in another preferred embodiment, where the first peptidecomprises a peptide from the RgpA[426-462] region, this peptide ispreferably selected from the RAS1[426-448], RAS2[427-462] andRAS3[430-449] peptides shown in Table 1. Alternatively, the firstpeptide may be the PAS1R[426-446] peptide, also known as PAS1(R45),disclosed in International Patent Application No. PCT/AU98/00311 (WO98/049192).

In the chimeric or fusion protein of the invention, the second peptidemay be a peptide from an adhesin domain of a P. gingivalis trypsin—likeenzyme, such as Lys-X-proteinase (Kgp) or Arg-X-proteinase (RgpA) orHagA (see Table 2). These domains are sometimes also known ashemagglutinins. In the Lys-X-proteinase, the preferred domains are KA1,KA2, KA3, KA4, KA5 as identified in Table 2. In the Arg-X-proteinase,the preferred domains are RA1, RA2, RA3 and RA4 as identified in Table2. In HagA, the preferred domains are HagA1, HagA1* and HagA1**.

TABLE 2 Adhesin domains of the Kgp and RgpA proteinases. A1 sA1 LA1 A2A3 A4 A5 Kgp Lys-X KA1 (738- KsA1 (759- KLA1 (751- KA2 (1157- KA3 (1292-KA4 (1427- KA5 (1548- proteinase 1099) 989) 1056) 1275) 1424) 1546)1732) SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. 62 NO:35 NO: 36 NO: 37 NO: 40 NO: 41 NO: 42 NO: 43 RgpA Arg-X RA1 (720- RsA1(831- — RA2 (1139- RA3 (1274- RA4 (1432- — proteinase 1081) 971) 1257)1404) 1706) SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. 61 NO: 38 NO:39 NO: 44 NO: 45 NO: 46 HagA HagA1 (26- SEQ ID 351) (SEQ NO. 63 ID NO:80), HagA1* (366-625) (SEQ ID NO: 81), HagA1** (820-1077) (SEQ ID NO:82) or HagA1** (1272-1529) (SEQ ID NO: 82)

In addition to improving the humoral response to a peptide of theinvention such as KAS1, KAS2, KAS3, KAS4, KAS5 and KAS6 or RAS1, RAS2and RAS3, RAS4 and RAS5 when included with such a peptide in a chimericor fusion protein, the adhesin domain also contains immunogenicepitopes, hence leading to the production of multiple specificities toelicit a protective immunogenic response. The finding that theimmunogenic epitopes of the adhesin domain are retained in a formapproaching that in a P. gingivalis trypsin—like enzyme when provided inthe chimeric or fusion protein of the invention is unanticipated.

It will be understood that in these embodiments of the invention thechimeric or fusion protein may contain any one or more of the peptidesselected from KAS1/RAS1, KAS2/RAS2, KAS3/RAS3, KAS4/RAS4, KAS5/RAS5 andKAS6/RAS6 together with any one or more adhesin domains of a P.gingivalis trypsin-like enzyme, in particular with any one or more ofLys-X-proteinase adhesin domains (KA1, KA2, KA3, KA4 and KA5) orArg-X-proteinase adhesin domains (RA1, RA2, RA3 and RA4) or HagA domainsHagA1, HagA1* and HagA1**.

It will also be understood that it is not necessary for the adhesindomain to be a complete domain as observed in a P. gingivalistrypsin-like enzyme. For example the adhesin domain may be a fragment ofsuch a domain, in particular, preferred fragments are the KsA1 and KLA1domain fragments of the Lys-X-proteinase A1 domain (see Table 2). Wherethe domain is a fragment of an adhesin domain it generally contains oneor more adhesin domain specific epitopes.

The sequence identifiers corresponding to the adhesin related peptidesare shown in Table 3.

In one embodiment the second peptide or polypeptide includes a sequenceshown in one or more of SEQ ID No: 69 to 79 or one or more of 83 to 85.

The chimeric or fusion protein of the present invention may also includeone or more additional peptides selected from the Kgp[432-468] region ofthe Lys-X-proteinase and/or one or more additional peptides selectedfrom the RgpA[426-462] region of the Arg-X-proteinase.

In preferred embodiments of the present invention, the chimeric orfusion protein includes one or more of KAS1, KAS2, KAS3, KAS4, KAS5 andKAS6, or one or more of RAS1, RAS2, RAS3, RAS4 and RAS5, together withKsA1 or KLA1.

Thus in certain embodiments, the chimeric or fusion protein may includeat least one further peptide wherein said further peptide includes:

-   -   (i) part of, or all of a sequence that is the same as, or        homologous to the sequence shown in SEQ ID No:1; or    -   (ii) part of, or all of a sequence that is the same as, or        homologous to the sequence shown in SEQ ID No:2; or    -   (iii) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Lys-X-proteinase of P. gingivalis; or    -   (iv) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Arg-X-proteinase of P. gingivalis; or    -   (v) part of, or all of a sequence that is the same as, or        homologous to the sequence of a HagA adhesin domain of P.        gingivalis.

Other examples of domains, units, sequences or regions that may beincluded in a chimeric or fusion protein as described herein includedomains for binding to receptors or ligands such as Fc binding regionsor Fc receptors, domains for improving half-life such as albumin ordomains for facilitating expression or purification of the chimeric orfusion protein.

In the chimeric or fusion proteins of the present invention, theC-terminal residue of the first peptide may be covalently linked to theN-terminal residue of an adhesin domain polypeptide, or the N-terminalresidue of the first peptide may be covalently linked to the C-terminalresidue of an adhesin domain polypeptide. In this arrangement, the firstpeptide and adhesin domain polypeptide, are said to be “directly linked”or “adjacent”.

In other embodiments, the chimeric or fusion protein includes a linkerfor linking the first peptide to an adhesin domain polypeptide. Thelinker may be any linker able to join a peptide to a polypeptide,including both amino acid and non-amino acid linkers. Preferably, thelinker is non-immunogenic. Suitable linkers may be up to 15 amino acidsin length, although less than five amino acids is preferred. The linkermay function to bring the first peptide and adhesin domain polypeptideinto a closer spatial arrangement than normally observed in a P.gingivalis trypsin-like enzyme. Alternatively, it may space the firstpeptide and adhesin domain polypeptide apart.

The chimeric or fusion proteins of the invention may be produced byrecombinant expression systems (such as recombinant DNA technology) orby chemical synthesis (such as solid phase peptide synthesis). Thesetechniques are well known in the art.

The heterologous or chimeric protein is particularly advantageousbecause it improves the humoral response obtained over that obtainedusing the first or second peptide components of the chimeric or fusionprotein alone.

The inventors have found that chimeric proteins including these peptideshave a number of utilities. For example, as described herein, someproduce a humoral response that is highly protective for treatment orprevention of bone loss as observed in chronic periodontitis. Thepeptides may also be used in a diagnostic assay wherein they can detector monitor specificities in an individual's serum, thereby indicatingwhether or not the individual is infected and if so, whether treatmentsare required or if provided, whether they have been effective.

In one embodiment, the chimeric or fusion protein induces a protectiveimmune response, typically a response that at least minimises or limitsconnective tissue damage otherwise associated with P. gingivalisinfection. In one embodiment the protective response at least minimisesor limits P. gingivalis induced bone loss. A model system for measuringbone loss mediated by P. gingivalis infection is discussed herein.Typically the protective immune response is predominantly a humoralresponse. In certain embodiments the protective immune response alsoincludes a cellular response.

The present invention also provides a composition including a chimericor fusion protein as broadly described above. Typically the compositionis antigenic or immunogenic. More particularly, the invention provides acomposition suitable for eliciting a protective or therapeutic immuneresponse against P. gingivalis infection, including the chimeric orfusion protein, optionally in association with an adjuvant. Such acomposition may also include another component for modulating orpotentiating the immune response. One embodiment, the composition takesthe form of a vaccine.

Various adjuvants are known for use in conjunction with vaccinecompositions. The adjuvants aid by modulating the immune response and inattaining a more durable and higher level of immunity using smalleramounts of vaccine antigen or fewer doses than if the vaccine antigenwere administered alone. Examples of adjuvants include incompleteFreund's adjuvant (IFA), Adjuvant 65 (containing peanut oil, mannidemonooleate and aluminium monostearate), oil emulsions, Ribi adjuvant,the pluronic polyols, polyamines, Avridine, Quil A, saponin, MPL, QS-21,mineral gels such as aluminium salts and calcium salts, nanoparticlessuch as hydroxyapatite, calcium phosphate, aluminium salts, sugaroligomers and polymers such as mannan, chitosan. Other examples includeoil in water emulsions such as SAF-1, SAF-0, MF59, Seppic ISA720, andother particulate adjuvants such ISCOMs™ and ISCOM matrix™. An extensivebut not exhaustive list of other examples of adjuvants are listed in Coxand Coulter 1992 [In: Wong WK (ed.) Animals parasite control utilisingtechnology. Bocca Raton; CRC press, 1992; 49-112]. In addition to theadjuvant, the vaccine composition may include conventionalpharmaceutically acceptable carriers, excipients, fillers, buffers ordiluents as appropriate. One or more doses of the vaccine compositioncontaining adjuvant may be administered prophylactically to preventperiodontitis or therapeutically to treat already present periodontitis.

In a preferred composition, the chimeric or fusion protein is combinedwith a mucosal adjuvant and administered via the oral, buccal or nasalroute. Examples of mucosal adjuvants are nanoparticles, cholera toxinand heat labile E. coli toxin, the non-toxic B subunits of these toxins,genetic mutants of these toxins which have a reduced toxicity. Othermethods which may be utilised to deliver the antigenic proteinorally/buccally/nasally include incorporation or absorption of theprotein into or onto particles of biodegradable polymer (such asacrylates or polyesters) or nanoparticles (such as hydroxyapatite) bymicroencapsulation to aid uptake of the microspheres from thegastrointestinal tract or other mucosal surfaces and to protectdegradation of the proteins. Liposomes, ISCOMs™, hydrogels are examplesof other potential methods which may be further enhanced by theincorporation of targeting molecules such as LTB, CTB or lectins fordelivery of the antigenic protein to the mucosal immune system. Inaddition to the antigenic protein and the mucosal adjuvant or deliverysystem, the vaccine composition may include conventionalpharmaceutically acceptable carriers, excipients, fillers, coatings,dispersion media, antibacterial or antifungal agents, and buffers ordiluents as appropriate.

In this aspect, the invention also provides a method of preventing orreducing the incidence or severity of a P. gingivalis—related conditionor disease in a subject, which comprises administering to the subject achimeric or fusion protein as described above, or an composition asdescribed above.

The subject may be a human or other animal subject, and is preferably ahuman.

Typically, the P. gingivalis-related condition or disease is chronicperiodontis, however it may also be bone loss, especially alveolar boneloss, or coronary artery disease.

Many methods are known for administration of a vaccine composition to ahuman or animal subject, including but not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,sub-lingual, buccal and oral administration. These routes ofadministration are particularly useful for vaccination.

In another aspect, the invention provides an antibody, preferably amonoclonal antibody, raised against a chimeric or fusion protein asbroadly described above.

These antibodies may be produced by standard techniques, and may be usedin passive immunisation of a subject. Accordingly, in this aspect, theinvention also provides a method of preventing or reducing the severityof a P. gingivalis-related disease or condition in a subject, whichcomprises administering to the subject an antibody as described above.

In a further aspect, the present invention provides a nucleic acidmolecule including a nucleotide sequence encoding a chimeric or fusionprotein as broadly described above, optionally operatively linked to atleast one regulatory element. In one embodiment the nucleic acid isprovided in isolated or substantially purified form.

The nucleic acid molecule may, for example, be inserted into a suitableexpression vector for production of the chimeric protein as arecombinant protein by insertion of the expression vector into aprokaryotic or eukaryotic host cell. Successful expression of therecombinant protein requires that the expression vector contains thenecessary regulatory elements for transcription and translation whichare compatible with, and recognised by the particular host cell systemused for expression. A variety of host cell systems may be utilized toexpress the recombinant protein, which include, but are not limited tobacteria transformed with a bacteriophage vector, plasmid vector, orcosmid DNA; yeast containing yeast vectors; fungi containing fungalvectors; insect cell lines infected with virus (e.g. baculovirus); andmammalian cell lines transfected with plasmid or viral expressionvectors, or infected with recombinant virus (e.g. vaccinia virus,adenovirus, adeno-associated virus, retrovirus, etc).

Using methods known in the art of molecular biology, various promotersand enhancers can be incorporated into the expression vector, toincrease the expression of the recombinant protein, provided that theincreased expression of the amino acid sequences is compatible with (forexample, non-toxic to) the particular host cell system used.

The selection of the promoter will depend on the expression system used.Promoters vary in strength, i.e. ability to facilitate transcription.Generally, it is desirable to use a strong promoter in order to obtain ahigh level of transcription of the coding nucleotide sequence andexpression into recombinant protein. For example, bacterial, phage, orplasmid promoters known in the art from which a high level oftranscription have been observed in a host cell system including E. coliinclude the lac promoter, trp promoter, recA promoter, ribosomal RNApromoter, the P_(R) and P_(L) promoters, lacUV5, ompF, bla, Ipp, and thelike, may be used to provide transcription of the inserted nucleotidesequence encoding amino acid sequences.

Other control elements for efficient transcription or translationinclude enhancers, and regulatory signals. Enhancer sequences are DNAelements that appear to increase transcriptional efficiency in a mannerrelatively independent of their position and orientation with respect toa nearby coding nucleotide sequence. Thus, depending on the host cellexpression vector system used, an enhancer may be placed either upstreamor downstream from the inserted coding sequences to increasetranscriptional efficiency. Other regulatory sites, such astranscription or translation initiation signals, can be used to regulatethe expression of the coding sequence.

In another embodiment, the vector may be a viral or bacterial vaccinevector, and used to provide a recombinant viral vaccine, a recombinantbacterial vaccine, a recombinant attenuated bacterial vaccine, or aninactivated recombinant viral vaccine. Vaccinia virus is the best knownexample, in the art, of an infectious virus that is engineered toexpress vaccine antigens derived from other organisms. The recombinantlive vaccinia virus, which is attenuated or otherwise treated so that itdoes not cause disease by itself, is used to immunize the host.Subsequent replication of the recombinant virus within the host providesa continual stimulation of the immune system with the vaccine antigensthereby providing long lasting immunity.

Other live vaccine vectors include: adenovirus, cytomegalovirus, andpreferably the poxviruses such as vaccinia [Paoletti and Panicali, U.S.Pat. No. 4,603,112] and attenuated Salmonella strains [Stocker et al.,U.S. Pat. Nos. 5,210,035; 4,837,151; and 4,735,801; and Curtiss et al.,1988, Vaccine 6:155-160]. Live vaccines are particularly advantageousbecause they continually stimulate the immune system which can confersubstantially long-lasting immunity. When the immune response isprotective against subsequent P. gingivalis infection, the live vaccineitself may be used in a preventive vaccine against P. gingivalis. Inparticular, the live vaccine can be based on a bacterium that is acommensal inhabitant of the oral cavity. This bacterium can betransformed with a vector carrying a recombinant chimeric protein andthen used to colonise the oral cavity, in particular the oral mucosa.Once colonised in the oral mucosa, the expression of the recombinantprotein will stimulate the mucosal associated lymphoid tissue to produceneutralising antibodies. To further illustrate this embodiment, usingmolecular biological techniques well known in the art, nucleotidesequences encoding the chimeric proteins of this invention may beinserted into the vaccinia virus genomic DNA at a site which allows forexpression of epitopes but does not negatively affect the growth orreplication of the vaccinia virus vector. The resultant recombinantvirus can be used as the immunogen in a vaccine formulation. The samemethods can be used to construct an inactivated recombinant viralvaccine formulation except that the recombinant virus is inactivated,such as by chemical means known in the art, prior to use as an immunogenand without substantially affecting the immunogenicity of the expressedimmunogen. The inactivated recombinant-vaccine may be formulated with asuitable adjuvant in order to enhance the immunological response to thevaccine antigens.

The invention also provides for the use of a nucleic acid moleculeincluding a nucleotide sequence encoding a chimeric or fusion protein ofthis invention directly as the vaccine formulation. Nucleotide sequencesencoding the chimeric proteins, operatively linked to one or moreregulatory elements, can be introduced directly to vaccinate anindividual (“direct gene transfer”) against pathogenic strains of P.gingivalis. Direct gene transfer into a vaccinated individual, resultingin expression of the genetic material by the vaccinated individual'scells such as vascular endothelial cells as well as the tissue of themajor organs, has been demonstrated by techniques in the art such as byinjecting intravenously an expression plasmid:cationic liposome complex[Zhu et al., 1993, Science 261:209-211]. Other effective methods fordelivering vector DNA into a target cell are known in the art. In oneexample, purified recombinant plasmid DNA containing viral genes hasbeen used to inoculate (whether parenterally, mucosally, or via gene-gunimmunization) vaccines to induce a protective immune response [Fynan etal. 1993, Proc Natl Acad Sci USA 90:11478-11482]. In another example,cells removed from an individual can be transfected or electroporated bystandard procedures known in the art, resulting in the introduction ofthe recombinant vector DNA intro the target cell. Cells containing therecombinant vector DNA may then be selected for using methods known inthe art, such as by use of a selection marker expressed in the vector,and the selected cells may then be re-introduced into the individual toexpress the recombinant protein.

In this aspect, the invention further provides a method of preventing orreducing the incidence or severity of a P. gingivalis-related conditionor disease in a subject, which comprises administering to the subject anucleic acid molecule as described above, a vector as described above,or a prokaryotic or eukaryotic cell as described above.

In other embodiments there is provided a pharmaceutical compositionincluding a chimeric or fusion protein or an antibody as describedabove. The composition may further include diluent, excipient, orcarrier or chemotherapeutic agent for treatment of a P.gingivalis-related condition or disease and may be adapted for oraladministration. The compositions of this invention may be incorporatedin lozenges, or in chewing gum or other products, e.g. by stirring intoa warm gum base or coating the outer surface of a gum base, illustrativeof which are jelutong, rubber latex, vinylite resins, etc., desirablywith conventional plasticizers or softeners, sugar or other sweetenersor such as glucose, sorbitol and the like.

An oral composition of this invention which contains the above-mentionedpharmaceutical composition may be prepared and used in various formsapplicable to the mouth such as dentifrice including toothpastes,toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums,dental pastes, gingival massage creams, gargle tablets, dairy productsand other foodstuffs. An oral composition according to this inventionmay further include additional well known ingredients depending on thetype and form of a particular oral composition.

In certain preferred forms of the invention the oral composition may besubstantially liquid in character, such as a mouthwash or rinse. In sucha preparation the vehicle is typically a water-alcohol mixture desirablyincluding a humectant as described below. Generally, the weight ratio ofwater to alcohol is in the range of from about 1:1 to about 20:1. Thetotal amount of water-alcohol mixture in this type of preparation istypically in the range of from about 70 to about 99.9% by weight of thepreparation. The alcohol is typically ethanol or isopropanol. Ethanol ispreferred.

The pH of such liquid and other preparations of the invention isgenerally in the range of from about 5 to about 9 and typically fromabout 5.0 to 7.0. The pH can be controlled with acid (e.g. citric acidor benzoic acid) or base (e.g. sodium hydroxide) or buffered (as withsodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogenphosphate, sodium dihydrogen phosphate, etc).

In other desirable forms of this invention, the pharmaceuticalcomposition may be substantially solid or pasty in character, such astoothpowder, a dental tablet or a toothpaste (dental cream) or geldentifrice. The vehicle of such solid or pasty oral preparationsgenerally contains dentally acceptable polishing material.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol and polypropyleneglycol exemplify suitable humectants/carriers. Also advantageous areliquid mixtures of water, glycerine and sorbitol. In clear gels wherethe refractive index is an important consideration, about 2.5−30% w/w ofwater, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitolare preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellosolve and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationswill usually be sold or otherwise distributed in suitable labelledpackages. Thus, a bottle of mouth rinse will have a label describing it,in substance, as a mouth rinse or mouthwash and having directions forits use; and a toothpaste, cream or gel will usually be in a collapsibletube, typically aluminium, lined lead or plastic, or other squeeze, pumpor pressurized dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents may be used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, non-ionic or ampholytic in nature and preferablydoes not interact with the active agent. It is preferred to employ asthe surface-active agent a detersive material which imparts to thecomposition detersive and foaming properties. Suitable examples ofanionic surfactants are water-soluble salts of higher fatty acidmonoglyceride monosulfates, such as the sodium salt of the monosulfatedmonoglyceride of hydrogenated coconut oil fatty acids, higher alkylsulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such assodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higherfatty acid esters of 1,2-dihydroxy propane sulfonate, and thesubstantially saturated higher aliphatic acyl amides of lower aliphaticamino carboxylic acid compounds, such as those having 12 to 16 carbonsin the fatty acid, alkyl or acyl radicals, and the like. Examples of thelast mentioned amides are N-lauroyl sarcosine, and the sodium,potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, orN-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. Examples of water-soluble non-ionicsurfactants suitable for use are condensation products of ethylene oxidewith various reactive hydrogen-containing compounds reactive therewithhaving long hydrophobic chains (e.g. aliphatic chains of about 12 to 20carbon atoms), which condensation products (“ethoxamers”) containhydrophilic polyoxyethylene moieties, such as condensation products ofpoly (ethylene oxide) with fatty acids, fatty alcohols, fatty amides,polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide(e.g. Pluronic materials).

The surface active agent is typically present in amount of about 0.1-5%by weight. It is noteworthy, that the surface active agent may assist inthe dissolving of the active agent of the invention and thereby diminishthe amount of solubilizing humectant needed.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavouring or sweetening material may also be employed.Examples of suitable flavouring constituents are flavouring oils, e.g.oil of spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavour and sweetening agents may each or together comprisefrom about 0.1% A to 5% more of the preparation.

Compositions intended for oral use may be prepared according to anymethod known in the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavouringagents, colouring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract or periodontal pocket and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.

The aqueous suspensions may also contain one or more preservatives orantimicrobial agents, for example benzoates, such as ethyl, or n-propylp-hydroxybenzoate another example is chlorhexidine gluconate, one ormore colouring agents, one or more flavouring agents, and one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide palatable oralpreparations. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

In a further aspect, the present invention provides a method for thediagnosis or monitoring of a P. gingivalis-related condition or diseasein a subject, which comprises use of a chimeric or fusion protein asdescribed above to detect anti-P. gingivalis antibodies in a biologicalsample from said subject.

In yet another aspect, the invention provides a method for the diagnosisor monitoring of a P. gingivalis-related condition or disease in asubject, which comprises use of an antibody as described above, todetect the presence of P. gingivalis in a biological sample from saidsubject.

In yet another aspect, the invention provides a peptide for inducing animmune response to P. gingivalis including the sequence shown in one ofSEQ ID No: 17, 18, 25 and 26. In one embodiment, the peptide has asequence that is homologous to one of SEQ ID No: 17, 18, 25 and 26. Thepeptide may have a length of 5 to 40 amino acids.

In yet another aspect, the invention provides a nucleic acid encoding apeptide having a sequence shown in one of SEQ ID No: 17, 18, 25 and 26.

In yet another aspect, the invention provides a use of a peptide havinga sequence shown in one of SEQ ID No: 17, 18, 25 and 26, or a nucleicacid encoding a peptide having a sequence shown in one of SEQ ID No: 17,18, 25 and 26, for the manufacture of a chimeric or fusion protein forinducing an immune response to P. gingivalis.

In yet another aspect, the invention provides a use of a peptide havinga sequence shown in one of SEQ ID No: 17, 18, 25 and 26, or a nucleicacid encoding a peptide having a sequence shown in one of SEQ ID No: 17,18, 25 and 26, for inducing an immune response to P. gingivalis. In oneembodiment, the peptide is administered simultaneously or sequentiallywith a second peptide including:

-   -   (i) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Lys-X-proteinase of P. gingivalis; or    -   (ii) part of, or all of a sequence that is the same as, or        homologous to the sequence of an adhesin domain of the        Arg-X-proteinase of P. gingivalis; or    -   (iii) part of, or all of a sequence that is the same as, or        homologous to the sequence of a HagA adhesin domain of P.        gingivalis.

TABLE 3 SEQ ID NO: Amino acid sequence Fragment  1LNTGV[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]T Kgp[432-[A/T][T/S]Q[V/L]KALTNK[D/N]K 468]  2FNGGISL[V/A]NYTGHGSETAWGTSHFGTTHVKQLTNSN RgpA[426- Q 462]  3 VSFANYT  4VGFANYT  5 GVSFANYT  6 GVGFANYT  7 VSFANYTA  8 VGFANYTA  9 ETAWAD 10ETSWAD 11 TAWADP 12 TSWADP 13 SETAWAD 14 SETSWAD 15 ETAWADP 16 ETSWADP17 TAWADPL 18 TSWADPL 19 GSETAWAD 20 GSETSWAD 21 SETAWADP 22 SETSWADP 23ETAWADPL 24 ETSWADPL 25 TAWADPLL 26 TSWADPLL 27LNTGV[G/S]FANYTAHGSET[S/A]WADP[S/L] KAS1 28NTGV[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]T KAS2[A/T][T/S]Q[V/L]KALTNK[D/N]K 29 V[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]KAS3 30 LNTGVSFANYTAHGSETAWADP PAS1K 31 FNGGISL[V/A]NYTGHGSETAWGTSH RAS132 NGGISL[V/A]NYTGHGSETAWGTSHFGTTHVKQLTNSNQ RAS2 33ISL[V/A]NYTGHGSETAWGTSHF RAS3 34 FNGGISLANYTGHGSETAWGT PAS1R 35ANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPATG KA1PLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGGKLTFWCAQDANYASEHYAVYASSTGNDASN FTNALLEETITA 36FLLDADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPAN KsA1ADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESF 37WGDNTGYQFLLDADHNTFGSVIPATGPLFTGTASSNLYS KLA1ANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGG 38SGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDT RA1HTLWPNCSVPANLFAPFEYTVPENADPSCSPTNMIMDGTASVNIPAGTYDFAIAAPQANAKIWIAGQGPTKEDDYVFEAGKKYHFLMKKMGSGDGTELTISEGGGSDYTYTVYRDGTKIKEGLTATTFEEDGVATGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGGKLTFWCAQDANYASEHYAVYASSTGNDA SNFTNALLEETITA 39DDYVFEAGKKYHFLMKKMGSGDGTELTISEGGGSDYTYT RsA1VYRDGTKIKEGLTATTFEEDGVATGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAP NGTPNPNPNPNPNPNPGTTTLSESF 40ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQ KA2LDWLTAHGGSNVVSSFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETP NGIN 41PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFT KA3MGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKKCVNVTVNSTQFNPVQNLTAEQ APNSMDAILKWNAPAS 42AEVLNEDFENGIPASWKTIDADGDGNNWTTTPPPGGSSF KA4AGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPGGGTLTFWVCAQDANYASEHYAVYASSTGNDASNFANALLEEVL TA 43TVVTAPEAIRGTRAQGTWYQKTVQLPAGTKYVAFRHFGC KA5TDFFWINLDDVVITSGNAPSYTYTIYRNNTQIASGVTETTYRDPDLATGFYTYGVKVVYPNGESAIETATLNITSLADVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGRNTVV YTAQGGHYAVMVVVDGKSYVEKLAVK 44ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQ RA2LDWLTAHGGTNVVSSFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETP NGIN 45PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFT RA3MGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKKCVNVTVNSTQFNPVKNLKAQP DGGDVVLKWEAPSA 46ANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPATG RA4PLFTGTASSDLYSANFESLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTETTYRDAGMSAQSHEYCVEVKYTAGVSPKVCVDYIPDGVADVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGRNTVVYTAQGGYYAVMVVVDGKSYVEKLAI K SEQ ID NO: Nucleotide sequence47 GACCATGGCTCATCACCATCACCATCACAATACCGG KAS2- AGTCAGCTTTGCA FOR 48GACTCGAGTTATTTGTCCTTATTAGTGAGTGCTTTC KAS2- REV 49GACCATGGCTTGGGGAGACAATACGGGTTAC KLA1- FOR 50 GACTCGAGACCTCCGTTAGGCAAATCCKLA1- REV 51 CCGTATTGTCTCCCCATTTGTCCTTATTAGTGAGTGC KAS2- TTTC KLA1- REV52 CACTAATAAGGACAAATGGGGAGACAATACGGGTTA KAS2- C KLA1- FOR 53CATGGATCTGAGACCGCATGGGCTGATCCACTTTTC KAS1- TTGTTGGATGCCGAT KsA1- FOR1 54CCATGGCTTTGAATACCGGAGTCAGCTTTGCAAACT KAS1- ATACAGCGCATGGATCTGAGACCGCAKsA1- FOR2 55 CTCGAGGAATGATTCGGAAAGTGTT KAS1- KsA1- REV 56CCATGGCTGATTATAGCTGGAATTCCCAGGTAGTCA multi- GCTTTGCAAACTATACA FOR1 57CTTTGCAAACTATACAGCGCATGGATCTGAGACCGC multi- ATGGGCTGATCCACTT FOR2 58ATGGGCTGATCCACTTCTGAATTCTTATTGGGGCGA multi- GATCGGCAATATTACC FOR3 59GATCGGCAATATTACCCATATTGGTGCTCATTACGC multi- TTGGGGAGACAATACG FOR4 60CTCGAGACCTCCGTTAGGCAAATCCAATGCCGGTGT multi-REV TATCAGATAGTTGTCA SEQ IDFull NO: Amino acid sequence length 61MKNLNKFVSIALCSSLLGGMAFAQQTELGRNPNVRLLES RgpATQQSVTKVQFRMDNLKFTEVQTPKGIGQVPTYTEGVNLSEKGMPTLPILSRSLAVSDTREMKVEVVSSKFIEKKNVLIAPSKGMIMRNEDPKKIPYVYGKTYSQNKFFPGEIATLDDPFILRDVRGQVVNFAPLQYNPVTKTLRIYTEITVAVSETSEQGKNILNKKGTFAGFEDTYKRMFMNYEPGRYTPVEEKQNGRMIVIVAKKYEGDIKDFVDWKNQRGLRTEVKVAEDIASPVTANAIQQFVKQEYEKEGNDLTYVLLIGDHKDIPAKITPGIKSDQVYGQIVGNDHYNEVFIGRFSCESKEDLKTQIDRTIHYERNITTEDKWLGQALCIASAEGGPSADNGESDIQHENVIANLLTQYGYTKIIKCYDPGVTPKNIIDAFNGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQLPFIFDVACVNGDFLFSMPCFAEALMRAQKDGKPTGTVAIIASTINQSWASPMRGQDEMNEILCEKHPNNIKRTFGGVTMNGMFAMVEKYKKDGEKMLDTWTVFGDPSLLVRTLVPTKMQVTAPAQINLTDASVNVSCDYNGAIATISANGKMFGSAVVENGTATINLTGLTNESTLTLTVVGYNKETVIKTINTNGEPNPYQPVSNLTATTQGQKVTLKWDAPSTKTNATTNTARSVDGIRELVLLSVSDAPELLRSGQAEIVLEAHDVWNDGSGYQILLDADHDQYGQVIPSDTHTLWPNCSVPANLFAPFEYTVPENADPSCSPTNMIMDGTASVNIPAGTYDFAIAAPQANAKIWIAGQGPTKEDDYVFEAGKKYHFLMKKMGSGDGTELTISEGGGSDYTYTVYRDGTKIKEGLTATTFEEDGVATGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNPNPNPNPNPNPGTTTLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGGKLTFWCAQDANYASEHYAVYASSTGNDASNFTNALLEETITAKGVRSPEAMRGRIQGTWRQKTVDLPAGTKYVAFRHFQSTDMFYIDLDEVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDWLTAHGGTNVVSSFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEADGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKKCVNVTVNSTQFNPVKNLKAQPDGGDVVLKWEAPSAKKTEGSREVKRIGDGLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPATGPLFTGTASSDLYSANFESLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTETTYRDAGMSAQSHEYCVEVKYTAGVSPKVCVDYIPDGVADVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGRNTVVYTAQGGYYAVMVVVDGKSYVEKLAIK 62MRKLLLLIAASLLGVGLYAQSAKIKLDAPTTRTTCTNNSF KgpKQFDASFSFNEVELTKVETKGGTFASVSIPGAFPTGEVGSPEVPAVRKLIAVPVGATPVVRVKSFTEQVYSLNQYGSEKLMPHQPSMSKSDDPEKVPFVYNAAAYARKGFVGQELTQVEMLGTMRGVRIAALTINPVQYDVVANQLKVRNNIEIEVSFQGADEVATQRLYDASFSPYFETAYKQLFNRDVYTDHGDLYNTPVRMLVVAGAKFKEALKPWLTWKAQKGFYLDVHYTDEAEVGTTNASIKAFIHKKYNDGLAASAAPVFLALVGDTDVISGEKGKKTKKVTDLYYSAVDGDYFPEMYTFRMSASSPEELTNIIDKVLMYEKATMPDKSYLEKVLLIAGADYSWNSQVGQPTIKYGMQYYYNQEHGYTDVYNYLKAPYTGCYSHLNTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKYFLAIGNCCITAQFDYVQPCFGEVITRVKEKGAYAYIGSSPNSYWGEDYYWSVGANAVFGVQPTFEGTSMGSYDATFLEDSYNTVNSIMWAGNLAATHAGNIGNITHIGAHYYWEAYHVLGDGSVMPYRAMPKTNTYTLPASLPQNQASYSIQASAGSYVAISKDGVLYGTGVANASGVATVSMTKQITENGNYDVVITRSNYLPVIKQIQVGEPSPYQPVSNLTATTQGQKVTLKWEAPSAKKAEGSREVKRIGDGLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLPNGGKLTFWCAQDANYASEHYAVYASSTGNDASNFTNALLEETITAKGVRSPKAIRGRIQGTWRQKTVDLPAGTKYVAFRHFQSTDMFYIDLDEVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDWLTAHGGSNVVSSFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKKCVNVTVNSTQFNPVQNLTAEQAPNSMDAILKWNAPASKRAEVLNEDFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPGGGTLTFWCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAPEAIRGTRAQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDVVITSGNAPSYTYTIYRNNTQIASGVTETTYRDPDLATGFYTYGVKVVYPNGESAIETATLNITSLADVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGR NTVVYTAQGGHYAVMVVVDGKSYVEKLAVK63 MRKLNSLFSLAVLLSLLCWGQTAAAQGGPKTAPSVTHQ HagAAVQKGIRTSKAKDLRDPIPAGMARIILEAHDVWEDGTGYQMLWDADHNQYGASIPEESFWFANGTIPAGLYDPFEYKVPVNADASFSPTNFVLDGTASADIPAGTYDYVIINPNPGIIYIVGEGVSKGNDYVVEAGKTYHFTVQRQGPGDAASVVVTGEGGNEFAPVQNLQWSVSGQTVTLTWQAPASDKRTYVLNESFDTQTLPNGWTMIDADGDGHNWLSTINVYNTATHTGDGAMFSKSWTASSGAKIDLSPDNYLVTPKFTVPENGKLSYWSSQEPWTNEHYGVFLSTTGNEAANFTIKLLEETLGSGKPAPMNLVKSEGVKAPAPYQERTIDLSAYAGQQVYLAFRHFGCTGIFRLYLDDVAVSGEGSSNDYTYTVYRDNVVIAQNLTATTFNQENVAPGQYNYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPNGGTLTFWCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAPEAIRGTRVQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLGWLTAHGGTNVVASFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKECVNVTVDPVQFNPVQNLTGSAVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKTIDADGDGNNWTTTPPPGGTSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPNGGTLTFWWCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAPEAIRGTRVQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDWLTAHGGTNVVASFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKECVNVTVDPVQFNPVQNLTGSAVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKTIDADGDGNNWTTTPPPGGTSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPNGGTLTFWCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAPEAIRGTRVQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDVEIKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLGWLTAHGGTNVVASFSWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYTAGVSPKECVNVTINPTQFNPVQNLTAEQAPNSMDAILKWNAPASKRAEVLNEDFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPGGGTLTFWCAQDANYASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAPEAIRGTRVQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWINLDDVVITSGNAPSYTYTIYRNNTQIASGVTETTYRDPDLATGFYTYGVKVVYPNGESAIETATLNITSLADVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGRNTVVYTAQGGHYAVMVVVDGK SYVEKLAVK SEQ ID NO:Amino acid sequence Fragment 64 D[S/Y][Y/S]WN[P/S][K/Q][W/V] KAS4 65NSYWGED KAS5 66 IGN[V/I]THIGAHY KAS6 67 EGGPSADN RAS4 68[N/D]Q[S/Y]WA[S/P]P RAS5 69 PVSNLTATTQGQKVTLKWDAPST ABM1 - RgpA_(cat) 70PVSNLTATTQGQKVTLKWEAPSA ABM1 - Kgpcat 71 PVQNLTGSSVGQKVTLKWDAPST ABM1 -KgpA1 72 PVQNLTGSAVGQKVTLKWDAPNG ABM1 - RgpA1 & RgpAA3 73PVKNLKAQPDGGDVVLKWEAPSA ABM1 - HagAA1*/ ** 74 PVQNLTAEQAPNSMDAILKWNAPABM1 - KgpA3 & HagAA3 75 PVQNLTQWSVSGQTVTLTWQAPAS ABM2 - HagAA1 76YTYTVYRDGTKIKEGLTETTFEEDGVA ABM2 - ABM2 - RgpAA4 77YTYTVYRDNVVIAQNLTATTFNQENVA ABM2 - HagA1* 78YTYTVYRDGTKIKEGLTA/ETTFEEDGVA ABM2 All other adhesins 79,PNGTP(NP)₁₋₆GTT(T)LSESF, wherein (T) may ABM3- All 110-or may not be present adhesins 114 80GGPKTAPSVTHQAVQKGIRTSKAKDLRDPIPAGMARIILE HagA1AHDVWEDGTGYQMLWDADHNQYGASIPEESFWFANGTI [26-351]PAGLYDPFEYKVPVNADASFSPTNFVLDGTASADIPAGTYDYVIINPNPGIIYIVGEGVSKGNDYVVEAGKTYHFTVQRQGPGDAASVVVTGEGGNEFAPVQNLQWSVSGQTVTLTWQAPASDKRTYVLNESFDTQTLPNGWTMIDADGDGHNWLSTINVYNTATHTGDGAMFSKSWTASSGAKIDLSPDNYLVTPKFTVPENGKLSYWSSQEPWTNEHYGVFLSTTGNEAA NFTIKLLEETLGSG 81APAPYQERTIDLSAYAGQQVYLAFRHFGCTGIFRLYLDDV HagA1*AVSGEGSSNDYTYTVYRDNVVIAQNLTATTFNQENVAPG [366-625]QYNYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPNGGTLTFWCAQDANYASE HYAVYASSTGNDASNFANALLEEVLTA 82PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFT HagA1**MGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGN [820-HEYCVEVKYTAGVSPKECVNVTVDPVQFNPVQNLTGSA 1077] orVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKT HagA1**IDADGDGNNWTTTPPPGGTSFAGHNSAICVSSASYINFE [1272-GPQNPDNYLVTPELSLPNGGTLTFWCAQDANYASEHY 1529] AVYASSTGNDASNFANALLEEVLTA 83PYQPVSNLTATTQGQ ABM1[436- 450] 84 EGLTATTFEEDGVAA ABM2 [672-686] 85GTPNPNPNPNPNPNPGT ABM3 [455-471]

The invention is further illustrated by the following Examples which areincluded by way of exemplification and not limitation of the invention.

Example 1

Methods and Materials.

Bacterial strains and growth conditions. Lyophilised cultures ofPorphyromonas gingivalis W50 were grown anaerobically at 37° C. on lysedhorse blood agar plates supplemented with 5 μg/ml haemin, 0.5 μg/mlcysteine (HB agar, <10 passages). After 3-4 days colonies were used toinoculate brain heart infusion medium containing 5 μg/ml haemin, 0.5μg/ml cysteine (1). Batch cultures were grown anaerobically in a MK3Anaerobic Workstation (Don Whitley Scientific Ltd., Adelaide,Australia). Cells were harvested during exponential growth phase bycentrifugation (7500 g, 30 min, 4° C.) and washed twice with PG buffer(50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂), and 5 mM cysteine-HCl, pH8.0) in the anaerobic workstation. Growth of batch cultures wasmonitored at 650 nm using a spectrophotometer (model 295E,Perkin-Elmer). Culture purity was checked routinely by Gram stain,microscopic examination and using a variety of biochemical testsaccording to Slots (2).

Construction of pET28 constructs containing adhesin sequences andadhesin sequences with N-terminal addition of Kgp proteinase sequences.Kgp residues representing peptides and chimeric peptides of the activesite (AS) and KgpA1 adhesin (A1) domains were over-expressed in E. colias recombinant (r) proteins with hexa-His tags (SEQ ID NO: 86) using pETexpression vectors (Novagen). The r-proteins expressed were rKAS2, andrKLA1 and the r-chimeric proteins were rKAS2-KLA1, rKAS1-KsA1 andrKAS4-KAS3-KAS5-KAS6-KLA1 (also referred to as multiKAS-KLA1). The aminoacid sequences representing the various A1 and AS domains are describedin Tables 1 and 2.

The various KAS and KA1 domains of the kgp gene were amplified from pNS1(3.5 kb BamHI lys fragment in pUC18) or P. gingivalis genomic DNArespectively using primers listed in Table 4, Taq DNA polymerase(Invitrogen) and a PC-960 thermal cycler (Corbett ResearchTechnologies). Primer pairs KAS2-FOR and KAS2-REV and KLA1-FOR andKLA1-REV were used to generate PCR fragments encoding KAS2 and KLA1respectively using the following reaction conditions: 94° C., 3 minutes,followed by 28 cycles of 94° C., 45 sec (denaturing); 62° C., 40 seconds(annealing) and 72° C., 20 seconds (extension) followed by a final cycleof 72° C., 5 min.

The KAS2-KLA1 chimeric PCR product was produced by gene splicing byoverlap extension (SOEing) as follows: PCR products were produced usingprimer pairs KAS2-FOR and KAS2-KLA1-chimera-REV andKAS2-KLA1-chimera-FOR and KLA1-REV using the conditions described above.The PCR products were then annealed and a final PCR was performed withprimers KAS2-FOR and KLA1-REV (94° C., 2 minutes, followed by 28 cyclesof 94° C., 30 sec; 50° C., 30 seconds and 72° C., 40 seconds followed bya final cycle of 72° C., 5 min.

For the preparation of the KAS1-KsA1 PCR product, two successive PCR'swere conducted using the KAS1-KsA1-REV primer with each of theKAS1-KsA1-FOR primers 1 and, 2 in succession (reaction conditions 94° C.for 2 minutes followed by 35 cycles of 94° C., 15 seconds; 63° C., 30seconds and 72° C., 2 minutes) to produce the KAS1-KsA1 PCR product. TheKAS1-KsA1-FOR1 and KAS1-KsA1-FOR2 primers contain an 3′extensionoverlapping the 5′ of the previous PCR product.

For the preparation of the multiKAS-KLA1 PCR fragment, four successivePCR's were conducted using the multi-REV primer with each of themulti-FOR primers 1, 2, 3 and 4 in succession (reaction conditions were95° C., 2 minutes followed by 35 cycles of 95° C., 20 seconds; 68° C.,1.5 minutes) to produce the multiKAS-KLA1 PCR product. Each multi-FORprimer contains a 3′extension overlapping the 5′ of the previous PCRproduct.

All of the PCR fragments encoding KAS2, KLA1, KAS2-KLA1, KAS1-KsA1 andmultiKAS-KLA1. were purified using PCR purification columns (Qiagen),ligated into the TA cloning vector, pGem-T Easy (Promega) andtransformed into E. coli JM109 following the manufacturer's protocol.Purified recombinant pGemT-Easy constructs were digested with Ncol andXhol and directionally cloned into Ncol/Xhol digested pET28b (Novagen)and transformed into the non-expression host, E. coli JM109 [DH5α]. Therecombinant pET28 constructs were purified and transformed into the E.coli expression host, BL21 (DE3) [HMS174(DE3)] (Novagen) and selected onLB containing 50 μg kanamycin following the manufacturer's instructions.The integrity of each insert was confirmed by DNA sequence analysis.

The oligonucleotide primers (Table 4) have been designed to incorporaterestriction enzyme sites, stop codons and hexa-His Tags (SEQ ID NO: 86)where necessary. The primers used for the rKAS2, rKLA1 and rKAS2-KLA1were designed to limit the inclusion of extraneous coding sequence to nomore than three amino acids plus the hexa-his tag (SEQ ID NO: 86) inr-proteins. The rKAS1 and the rKLA1 were designed to contain a hexa-Histag (SEQ ID NO: 86) at the N-terminal and C-terminal ends respectively,so that they may be directly compared to the rKAS2-KLA1 which has ahexa-his tag (SEQ ID NO: 86) at both N- and C-termini. In rKAS1-KsA1 andrmultiKAS-KLA1 the His Tags are found at the C-termini.

TABLE 4Oligonucleotide primers used for the amplification of the nucleotidesequences encoding the various fragments and chimeras of Kgp A1 and ASRecombinant (r) protein Oli Sequence (5′-3′) Characteristics* (5′-3′)rKAS2 KAS2- GACCATGGCTCATCACCATCACC GA buffer-Ncol (including ATG FORATCACAATACCGGAGTCAGCTTT start)-CT-(His)6-AS (nt 1992- GCA2012), “(His)6” disclosed as (SEQ ID NO: 47) SEQ ID NO: 86 KAS2-GACTCGAGTTATTTGTCCTTATTA GA buffer-Xhol-TTA Stop-KAS1 REV GTGAGTGCTTTC(nt 2099-2075) (SEQ ID NO: 48) rKLA1 KLA1- GACCATGGCTTGGGGAGACAATAGA buffer-Ncol (including ATG FOR CGGGTTAC (SEQ ID NO: 49)start)-CT-A1 (nt 2946-2966) KLA1- GACTCGAGACCTCCGTTAGGCAAGA buffer-Xhol-A1 (nt 3863- REV ATCC (SEQ ID NO: 50) 3845) rKAS2-KLA1KAS2- CCGTATTGTCTCCCCATTTGTCCT A1 (nt 2961-2946)-KAS1 (nt KLA1-TATTAGTGAGTGCTTTC 2099-2075) REV (SEQ ID NO: 51) KAS2-CACTAATAAGGACAAATGGGGAG KAS1 (nt 2084-2099)-A1 (nt KLA1- ACAATACGGGTTAC2946-2966) FOR (SEQ ID NO: 52) rKAS1-KsA1 KAS1- CATGGATCTGAGACCGCATGGGAS (nt 2025-2057)-A1 (nt 2970- KsA1- CTGATCCACTTTTCTTGTTGGATG 2987)-FOR1 CCGAT (SEQ ID NO: 53) KAS1- CCATGGCTTTGAATACCGGAGTCNcol-CT-AS (nt 1989-2042) KsA1- AGCTTTGCAAACTATACAGCGCA FOR2TGGATCTGAGACCGCA SEQ ID NO: 54) KAS1- CTCGAGGAATGATTCGGAAAGTGXhol-A1 (nt 3663-3644) KsA1- TT (SEQ ID NO: 55) REV rmultiKAS- multi-CCATGGCTGATTATAGCTGGAAT Ncol-CT-KAS4 (nt 1857-1880)- KLA1 FOR1TCCCAGGTAGTCAGCTTTGCAAA KAS3 (nt 2001-2021) CTATACA (SEQ ID NO: 56)multi- CTTTGCAAACTATACAGCGCATG KAS3 (nt 2006-2057) FOR2GATCTGAGACCGCATGGGCTGAT CCACTT (SEQ ID NO: 57) multi-ATGGGCTGATCCACTTCTGAATT KAS3 (nt 2042-2060)-KAS5 (nt FOR3CTTATTGGGGCGAGATCGGCAAT 2223-2240)-KAS6 (nt 2403- ATTACC (SEQ ID NO: 58)2417) multi- GATCGGCAATATTACCCATATTG G-KAS6 (nt 2403-2435)-GCT FOR4GTGCTCATTACGCTTGGGGAGAC (Ala spacer)-A1 (nt 2946-2960) AATACG(SEQ ID NO: 59) multi- CTCGAGACCTCCGTTAGGCAAAT Xho-A1 (nt 3863-3818) REVCCAATGCCGGTGTTATCAGATAG TTGTCA (SEQ ID NO: 60) *nucleotide (nt) sequencenumbers from lysine-specific cysteine proteinase gene sequence accessionnumber U75366

Expression and purification of recombinant proteins. Recombinantproteins were expressed from pET28::KLA1(KAS2, KAS2-LA1, KAS1-SA1,multiKAS-KLA1) constructs by induction with isopropylβ-D-thiogalactosidase (IPTG). All recombinant proteins were produced as6-His Tag (SEQ ID NO: 86) fusion proteins and purified with NI-NTApurification system (Invitrogen) under denaturing conditions. Briefly,E. coli (DE3) single colony transformants were used to inoculate 20 mLof Luria-Bertani (LB) broth containing 50 μg/ml kanamycin at 37° C. onan orbital shaker overnight. This inoculum was then used to inoculate 1L of LB containing 50 μg/ml kanamycin. The OD₆₀₀ of this culture wasallowed to reach 0.5-0.7 (mid-log phase) before inducing proteinexpression with isopropyl IPTG at 0.1 mM for 2 hours at 37° C. withshaking of 200 rpm. Cells were harvested (7,500 g) and resuspended in adenaturing binding buffer (8M Urea, 20 mM Sodium Phosphate pH 8.0 & 500mM NaCl) and sonicated on ice for 3×15 s bursts at 30 s intervals usinga Branson Sonifer 250 Cell disrupter (Branson Ultronics Corporation,Danbury, CT) with the microtip on setting 3, then centrifuged at 39,000g for 30 min at 4° C. Recombinant proteins were purified from thesupernatant by loading onto a pre-equilibrated Ni-NTA Agarose column andthen washing with denaturing washing buffer (8M Urea, 20 mM SodiumPhosphate pH 6.0 & 500 mM NaCl) to elute unbound proteins. The columnwas then washed using 10 volumes of binding buffer B and the recombinantprotein was eluted with denaturing elution buffer (8M Urea, 20 mM SodiumPhosphate pH 6.0, 500 mM NaCl & 0.5 M Imidazole). Purified protein wasdialyzed against 2M Urea-PBS and stored at −80° C.

Recombinant protein samples were analysed by SDS-PAGE and theirmolecular masses determined using ProtParam on-line(au.expasy.orq/tools/protparam.html). Protein concentration of allsamples was determined by the Bio-Rad Protein Assay using BSA as astandard.

Immunisation and the mouse periodontitis model. The mouse periodontitisexperiments were performed as described previously (3) and were approvedby the University of Melbourne Ethics Committee for AnimalExperimentation. BALB/c mice 6-8 weeks old (12 mice per group) housed inmicroisolators were immunized subcutaneously (s.c. 100 μL) with either50 μg of one of the recombinant proteins or RgpA-Kgp complex, 2×10⁹formalin killed cells of P. gingivalis strain W50 or PBS; each antigenwas emulsified in incomplete Freund's adjuvant (IFA). After 30 days themice were boosted with antigen (s.c. injection, emulsified in IFA) andthen bled from the retrobulbar plexus 12 days later. Four days after thesecond immunisation mice were given kanamycin (Sigma-Aldrich, New SouthWales, Australia) at 1 mg/ml in deionized water ad libitum for 7 days.Three days after the antibiotic treatment (2 days after bleeding), micewere orally inoculated four times 2 days apart with 1×10¹⁰ viable P.gingivalis W50 (25 μl) in PG buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mMCaCl₂), and 5 mM cysteine-HCl, pH 8.0) containing 2% (wt/vol)carboxymethyl cellulose (CMC; Sigma-Aldrich, New South Wales,Australia), and a control group was sham infected with PG buffercontaining 2% (wt/vol) CMC alone. The inocula were prepared in theanaerobic chamber and then immediately applied to the gingival margin ofthe maxillary molar teeth. Two weeks later, mice received another fourdoses (2 days apart) of 1×10¹⁰ cells of viable P. gingivalis W50 (25 μl)in PG buffer containing 2% (wt/vol) CMC. The number of viable bacteriain each inoculum was verified by enumeration on blood agar. Mice werefed a soft powdered diet (Barastock, Australia) and housed in cagesfitted with a raised wire mesh bottom to prevent access to bedding. Fourweeks after the last dose, mice were bled from the retrobulbar plexusand killed, and the maxillae were removed and cut in half with one half(right) used for alveolar bone loss measurement and the other half(left) used for real-time PCR.

The right half maxillae were boiled (1 min) in deionized water,mechanically defleshed, and immersed in 2% (wt/vol) potassium hydroxide(16 h, 25° C.). The half maxillae were then washed (two times withdeionized water) and immersed in 3% (wt/vol) hydrogen peroxide (6 h, 25°C.). After the half maxillae were washed (two times with deionizedwater), they were stained with 0.1% (wt/vol) aqueous methylene blue, anda digital image of the buccal aspect of each half maxilla was capturedwith an Olympus DP12 digital camera mounted on a dissecting microscope,using OLYSIA BioReport software version 3.2 (Olympus Australia Pty Ltd.,New South Wales, Australia) to assess horizontal bone loss. Horizontalbone loss is loss occurring in a horizontal plane, perpendicular to thealveolar bone crest (ABC) that results in a reduction of the crestheight. Each half maxilla was aligned so that the molar buccal andlingual cusps of each tooth image were superimposed, and the image wascaptured with a micrometer scale in frame, so that measurements could bestandardized for each image. The area from the cementoenamel junction tothe ABC for each molar tooth was measured using OLYSIA BioReportsoftware version 3.2 imaging software. Bone loss measurements weredetermined twice by a single examiner using a randomized and blindedprotocol.

Determination of subclass antibody by an ELISA. To determine thesubclass antibody responses of mouse sera, enzyme-linked immunosorbentassays (ELISAs) were performed in triplicate using a 5-μg/ml solution offormalin killed P. gingivalis W50 in phosphate-buffered saline (PBS)(0.01 M Na₂HPO₄, 1.5 mM KH₂PO₄, 0.15 M NaCl), pH 7.0, containing 0.1% A(vol/vol) Tween 20 (PBST) to coat wells of flat-bottom polyvinylmicrotiter plates (Dynatech Laboratories, McLean, VA). After removal ofthe coating solution, PBST containing 2% (wt/vol) skim milk powder wasadded to wells to block the uncoated plastic for 1 h at roomtemperature. After the wells were washed four times with PBST, serialdilutions of mouse sera in PBST containing 0.5% (wt/vol) skim milk(SK-PBST) were added to each well and incubated for 16 h at roomtemperature. After the wells were washed six times with PBST, a 1/2,000dilution of goat IgG to mouse IgM, IgA, IgG1, IgG2a, IgG2b, or IgG3(Sigma, New South Wales, Australia) was added in SK-PBST and allowed tobind for 2 h at room temperature. Plates were washed six times in PBST,and a 1/5,000 dilution of horseradish peroxidase-conjugated rabbitanti-goat immunoglobulin (Sigma, New South Wales, Australia) in SK-PBSTwas added to each well and incubated for 1 h at room temperature. Afterthe wells were washed six times with PBST, bound antibody was detectedby the addition of 100 μl of ABTS substrate [0.9 mM2,2′-azino-bis(3-ethylbenz-thiazoline-6) sulfonic acid in 80 mM citricacid containing 0.005% (vol/vol) hydrogen peroxide, pH 4.0] to eachwell. The optical density at 415 nm was measured using a microplatereader (Bio-Rad microplate reader, model 450).

SDS-PAGE gel electrophoresis and Western blotting. Recombinant proteins(10 μg) were analysed using the XCell surelock Mini-Cell electrophoresissystem. Recombinant proteins were mixed in 20 μl of reducing samplebuffer (10% [wt/vol] SDS, 0.05% [wt/vol] bromophenol blue, 25% [vol/vol]glycerol, and 0.05% [vol/vol] 2-mercaptoethanol). The pH was adjusted topH 8.0 with 1.5 M Tris-HCl, and then the solution was heated for 5 minat 100° C. Recombinant proteins (10 μg/lane) were loaded onto Novex 12%(wt/vol) Tris-glycine precast mini gels, and electrophoresis wasperformed using a current of 30 to 50 mA and a potential difference of125 V using a Novex electrophoresis system (Novex, San Diego, CA).Proteins were visualized using 0.25% w/v Coomassie blue R250.

Epitope analysis of the Kgp proteinase active site peptide (KAS-2)sequence. The antibody binding sites for the Lys-specific proteinaseactive site peptide KAS2 (433-468 SEQ ID No: 28) was determined bysynthesising N-terminally biotinylated overlapping eight residuepeptides (offset by one, overlapping by seven residues) on a multipinpeptide synthesis system (Chiron Technologies, Melbourne, Australia)using standard solid-phase peptide synthesis protocols for Fmocchemistry. Biotinylated peptides (5 μg/mL) in 0.1 M PBS, pH 7.4 werebound to strepavidin coated plates, overnight at 4° C. (Nunc, NSWAustralia). After the wells were washed four times with PBST epitopemapping of the plate-bound peptides was carried out by ELISA as perChiron Technologies instructions using mouse sera at a dilution of1:1000 in 1% w/v non-fat skim milk powder in 0.1 M PBS, pH 7.4,containing 0.1% v/v Tween 20 (SK-PBST). After the wells were washed sixtimes with PBST, a 1/2,000 dilution of goat IgG to mouse IgG (Sigma, NewSouth Wales, Australia) was added in SK-PBST and allowed to bind for 2 hat room temperature. Plates were washed six times in PBST, and a 1/5,000dilution of horseradish peroxidase-conjugated rabbit anti-goatimmunoglobulin (Sigma, New South Wales, Australia) in SK-PBST was addedto each well and incubated for 1 h at room temperature. After the wellswere washed six times with PBST, bound antibody was detected by theaddition of 100 μl of ABTS substrate [0.9 mM2,2′-azino-bis(3-ethylbenz-thiazoline-6) sulfonic acid in 80 mM citricacid containing 0.005% (vol/vol) hydrogen peroxide, pH 4.0] to eachwell. The optical density at 415 nm was measured using a microplatereader (Bio-Rad microplate reader, model 450).

Statistical analysis. The bone loss data were statistically analyzedusing a one-way analysis of variance (ANOVA) and Dunnett's T3 test (SPSSfor Windows, version 12). The IgA, IgM, and IgG subclass antibody titerswere statistically analyzed using Student's t test using SPSS software(SPSS for Windows, version 12).

Example 2

Characterisation and purification of the recombinant proteins (KsA1,KLA1, KAS1-KsA1 and KAS2-KLA1). In order to characterise the ability ofKgp adhesin A1 domain fragments and chimera Kgp proteinase and Kgpadhesin A1 domain fragments to protect against P. gingivalis infection,we expressed and purified the recombinant proteins:-KsA1, KLA1,KAS1-KsA1 and KAS2-KLA1. Recombinant proteins (KsA1 and KLA1) andrecombinant chimera proteins (KAS1-KsA1 and KAS2-KLA1) were purifiedfrom inclusion bodies using nickel chelate affinity chromatography andthe purified proteins analysed by SDS-PAGE (FIG. 1 ). Each of thepurified recombinant proteins consisted of one major protein band withmolecular weights of 40, 36, 31 and 32 kDa corresponding to KAS2-KLA1,KLA1, KsA1 and KAS1-KsA1, and these weights corresponded to thecalculated molecular masses of the His-tag recombinant proteins usingProtParam. To characterize the immunogenicity of the recombinantproteins KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1 were used to immunize miceand the sera was used to probe KAS2 peptide coated plates and formalinkilled P. gingivalis W50 cells coated plates (FIG. 2 ). Recombinantchimera proteins KAS1-KsA1 and KAS2-KLA1 antisera were found torecognize KAS2 peptide (FIG. 2A) at a similar level to KAS2 specificantisera (KAS2-diptheria toxoid conjugate) as well as formalin killed P.gingivalis W50 cells (FIG. 2B). However, antisera against therecombinant protein KLA1 only recognized killed P. gingivalis W50 cells(FIG. 2B).

Example 3

Effect of immunization with the recombinant proteins (KsA1, KLA1,KAS1-KsA1 and KAS2-KLA1) on P. gingivalis induced alveolar bone loss inthe mouse periodontitis model. The recombinant proteins KsA1, KLA1,KAS1-KsA1 and KAS2-KLA1, formalin killed P. gingivalis strain W50 andthe RgpA-Kgp complex were used to determine and compare the protectioninduced against P. gingivalis induced alveolar bone loss using amodified mouse model of periodontal bone loss based on that reported byBaker et al (4). Mice were immunized (days 0 and 30) with eitherrecombinant proteins KsA1, KLA1, KAS1-KsA1 or KAS2-KLA1, RgpA-Kgpcomplex or formalin killed P. gingivalis strain W50 (FK-W50) cells orPBS adjuvant alone and were then orally challenged with viable P.gingivalis W50. Immunization with all of the recombinant antigens,RgpA-Kgp complex and FK-W50 cells protected BALB/c mice against P.gingivalis-induced alveolar bone loss as these animals exhibitedsignificantly (p<0.001) less bone loss compared to the PBS immunizedgroup (FIG. 3 ). However the KAS2-KLA1 immunised mice had significantlyless bone loss than mice immunised with KLA1 (p<0.01); KsA1 (p<0.001),RgpA-Kgp complex (p<0.001), FK-W50 cells (p<0.001) and non-challengedmice (p<0.001). There was no significant difference in bone loss betweenthe KAS2-KLA1 and KAS1-KsA1 immunised mice. Furthermore, KAS1-KsA1immunised mice exhibited significantly less bone loss thannon-challenged mice (p<0.01) and RgpA-Kgp complex immunised mice(p<0.05), but were not significantly different from KsA1, KLA1, andFK-W50 immunised mice. There was no significant difference in bone lossbetween the KsA1, KLA1, RgpA-Kgp complex and FK-W50 immunised mice.

Example 4

Antibody subclass responses induced by immunization with the recombinantproteins (KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1) in the mouseperiodontitis models. Prior and post to oral inoculation challenge withviable P. gingivalis cells mice were bled and the sera collected bycentrifugation. FIG. 4 shows the antibody subclass reactivity toformalin-killed P. gingivalis W50 cells for each immunogen (KsA1, KLA1,KAS1-KsA1 or KAS2-KLA1 or formalin killed P. gingivalis strain W50(FK-W50) cells) in the mouse periodontitis model. All of the protectiveimmunogens induced a high IgG antibody titre to FK-W50. Furthermore, thepredominant antibody subclass each protective immunogen induced was IgG1with only weakly immunoreactive IgG2a, IgG2b and IgG3 FK-W50-specificantibodies (FIG. 4 ). The predominant antibody subclass induced by eachimmunogen both pre (FIG. 4A) and post—oral inoculation (FIG. 4B) wasIgG1.

Example 5

Epitope mapping of KAS2 (433-468). Overlapping biotinylated eightresidue peptides (offset by one, overlap by seven) for KAS2 (433-468)were synthesised and used to coat streptavidin coated plates. Theantibody binding epitopes were then identified using antisera from miceimmunized with KAS1-KsA1, KAS2-KLA1 and KAS2-diphtheria toxoid conjugate(FIG. 5 ). A two fold increase in optical density (415 nm) abovebackground was considered as a positive antibody response (thresholdOD). The antisera recognised the following peptide sequences derivedfrom SEQ ID No. 28 viz. KAS1-KsA1 recognised peptides 435-442, 436-443,445-452, 446-453 and 447-454 (threshold OD=0.07, FIG. 5A) whereasKAS2-KLA1 recognised peptides 435-442, 447-454 and 448-455 (thresholdID=0.07, FIG. 5A). This suggests recognition of a number of minimalepitopes viz. peptide 436-442 (VSFANYT (SEQ ID NO: 3) and its variantVGFANYT (SEQ ID NO: 4)), peptide 447-452 (ETAWAD (SEQ ID NO: 9) and itsvariant ETSWAD (SEQ ID NO: 10)), and peptide 448-453 (TAWADP (SEQ ID NO:11) and its variant TSWADP (SEQ ID NO: 12)). Peptides which include thepeptide 436-442 epitope include GVSFANYT (SEQ ID NO: 5), GVGFANYT (SEQID NO: 6), VSFANYTA (SEQ ID NO: 7) and VGFANYTA (SEQ ID NO: 8). Peptideswhich include the peptide 447-452 and/or 448-453 epitopes includeSETAWAD (SEQ ID NO: 13), SETSWAD (SEQ ID NO: 14), ETAWADP (SEQ ID NO:15), ETSWADP (SEQ ID NO: 16), TAWADPL (SEQ ID NO: 17) and TSWADPL (SEQID NO: 18), more particularly GSETAWAD (SEQ ID NO: 19), GSETSWAD (SEQ IDNO: 20), SETAWADP (SEQ ID NO: 21), SETSWADP (SEQ ID NO: 22), ETAWADPL(SEQ ID NO: 23), ETSWADPL (SEQ ID NO: 24), TAWADPLL (SEQ ID NO: 25) andTSWADPLL (SEQ ID NO: 26).

Example 6

Synthesis of KAS and RAS Peptides for Conjugation to a Protein.

Peptides were synthesized manually or using a CEM Microwave peptidesynthesizer. Standard solid-phase peptide synthesis protocols for Fmocchemistry were used throughout. Peptides were assembled as thecarboxyamide form using Rink-linker derived AM-sure resin (AAPPTEC, KY,USA). Coupling was accomplished with HBTU/HOBt activation using 4 equivof Fmoc-amino acid and 6 equiv of DIPEA. The Fmoc group was removed by20% piperidine in 1M HOBt/DMF.

Resins bearing KAS or RAS peptides were swollen in DMF and theN-terminal Fmoc group removed by 2% v/v DBU in DMF containing 2% v/vpiperidine. The N-terminal amino group was then derivatised withS-Acetylmercaptoacetic acid (SAMA) group using 5 equiv of SAMA-OPfp and5 equiv of HOBt. The reaction was monitored by the trinitrobenzenesulphonic acid (TNBSA) test. When a negative TNBSA test was returned theresin was washed (5×DMF, 3×DCM and 3×diethyl ether). The resin was thendried under vacuum. Cleavage of peptides from the resin support wasperformed using TFA:phenol:TIPS:EDT:water (92:2:2:2:2) cleavage cocktailfor 2.5 hours or 4 hours depending on the arginine content of thepeptide. After cleavage the resin was removed by filtration and thefiltrate concentrated to approximately 1 mL under a stream of nitrogen.After the peptide products were precipitated in cold ether, they werecentrifuged and washed three times. The peptide precipitates weredissolved in 5 to 10 mL of water containing 0.1% v/v TFA and insolubleresidue removed by centrifugation. Peptides were purified by RP-HPLC.

A number of different chemical moieties can be used for derivatisingpeptides for conjugation to proteins, these would introduced reactivegroups such as; halides (bromo, chloro and iodo), maleimido,succinimidyl, hydrazinyl, oxime, thiol, which would then be usedconjugate the derivatised peptide to a protein such as KgpA1 through itsnative cysteine residues or has been derivatised with the complementaryreactive group that allows the chemical ligation to proceed to form apeptide-protein conjugate.

Conjugation of SAMA-Peptides to KA1. To a solution, containing 10 mg/mLof recombinant KA1 or other adhesin domain of the RgpA-Kgp complex inphosphate-buffered saline (0.1M sodium phosphate, 0.9% NaCl, pH 7.4) wasadded 0.1 mL of a 1% w/v solution of m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) in DMF. After 30 min unreactedMBS was removed and MBS-modified KA1 collected by gel filtration using aPD10 column (Pharmacia, NSW, Australia) equilibrated in conjugationbuffer (0.1M sodium phosphate, 5 mM EDTA; pH 6.0). Purified SAMA-peptide(1.3 μmole) was dissolved in 200 μM guanidine HCl containing 0.5 M Tris;2 mM EDTA, pH 6.0 and diluted with 8004 MilliQ water and deprotectedin-situ by addition of 254 of 2M NH₂OH (40 equiv) dissolved in MilliQwater. The collected MBS-KA1 was immediately reacted with deprotectedSAMA-peptide and stirred for one hour at room temperature. Thepeptide-KA1 conjugate was separated from unreacted peptide by gelfiltration using a PD10 column equilibrated in PBS pH 7.4 andlyophilized. The reaction was monitored using the Ellmans test.

Example 7

Preparation of Antibodies. Polyclonal antiserum to recombinant proteinsare raised in mice by immunising with the proteins subcutaneously. Themice are immunised at day 0 with 25 μg of protein in incomplete Freund'sadjuvant and day 30 with 25 μg of protein in incomplete Freund'sadjuvant. Immunisations are carried out using standard procedures.Polyclonal antisera having a high titre against the proteins areobtained. If desired monoclonal antibodies directed specifically againstrecombinant proteins are obtained using standard procedures.

Example 8

Immunization for the generation of antibodies. BALB/c mice or CD1 (Swissout bred mices) 6-8 weeks old (10 mice per group) were immunizedsubcutaneously (s.c. 100 μL) with either 50 μg of the KAS2-LA1 chimeraand the antigen emulsified in incomplete Freund's adjuvant (IFA). After30 days the mice were boosted with antigen (s.c. injection, emulsifiedin IFA) and 12 days later the mice were killed and cardiac bled tocollect sera.

Determination of subclass antibody by an ELISA. To determine thesubclass antibody responses of mouse sera, enzyme-linked immunosorbentassays (ELISAs) were performed in triplicate using a 5-μg/ml solution ofKAS2-LA1 chimera or formalin killed P. gingivalis W50 or the RgpA-Kgpcomplex in phosphate-buffered saline (PBS) (0.01 M Na₂HPO₄, 1.5 mMKH₂PO₄, 0.15 M NaCl), pH 7.0, containing 0.1% (vol/vol) Tween 20 (PBST)to coat wells of flat-bottom polyvinyl microtiter plates (DynatechLaboratories, McLean, VA). After removal of the coating solution, PBSTcontaining 2% (wt/vol) skim milk powder was added to wells to block theuncoated plastic for 1 h at room temperature. After the wells werewashed four times with PBST, serial dilutions of mouse sera in PBSTcontaining 0.5% (wt/vol) skim milk (SK-PBST) were added to each well andincubated for 16 h at room temperature. After the wells were washed sixtimes with PBST, a 1/2,000 dilution of goat IgG to mouse IgM, IgA, IgG1,IgG2a, IgG2b, or IgG3 (Sigma, New South Wales, Australia) was added inSK-PBST and allowed to bind for 2 h at room temperature. Plates werewashed six times in PBST, and a 1/5,000 dilution of horseradishperoxidase-conjugated rabbit anti-goat immunoglobulin (Sigma, New SouthWales, Australia) in SK-PBST was added to each well and incubated for 1h at room temperature. After the wells were washed six times with PBST,bound antibody was detected by the addition of 100 μl of ABTS substrate[0.9 mM 2,2′-azino-bis(3-ethylbenz-thiazoline-6) sulfonic acid in 80 mMcitric acid containing 0.005% (vol/vol) hydrogen peroxide, pH 4.0] toeach well. The optical density at 415 nm was measured using a microplatereader (Bio-Rad microplate reader, model 450).

Antibody subclass responses induced by immunization with the recombinantprotein KAS2-KLA1 in outbred (CD1, Swiss) mice. CD1 (Swiss) mice wereimmunised with the KAS2-LA1 chimera, bled and the sera collected bycentrifugation. FIG. 6 shows the antibody subclass reactivity toKAS2-LA1 chimera, formalin-killed P. gingivalis W50 cells and theRgpA-Kgp complex. The KAS2-LA1 chimera induced a strong IgG antibodywith a predominant IgG1 antibody response that recognised the KAS2-LA1chimera and cross reacted strongly with FK P. gingivalis W50 cells andthe RgpA-Kgp complex (FIG. 6 ). Furthermore, the KAS2-LA1 chimerainduced only weak immunoreactive IgG2a, IgG2b and IgG3 antigen-specificantibodies (FIG. 6 ).

Example 9

Development of a Kgp structural model and Identification of Active SiteSurface Accessible Sequences.

Our work has shown that Kgp proteinase active site peptides are highlyimmunogenic and induce high levels of protection against P.gingivalis-induced bone loss. In an attempt to identify furtherproteinase active site peptides as vaccine candidates a model of thecatalytic domain of Kgp was developed using the Orchestrar suite ofprograms within Sybyl7.3 (FIG. 7 ). The model is based on PDB structure1crv of the RgpB protease from P. gingivalis, the proteins have a 23.58%pairwise identity and the Z-score is 25.09 (a high-confidence model).The Meta-PPisp protein interaction server predicts two protein-proteininteraction surfaces for Kgp: the substrate binding surface (as inRgpB), and a second surface unique to Kgp. The major differences betweenthe RgpB and Kgp models are in the loops that frame the secondinteraction surface and a 19-residue gap (Va1526 to Phe545) thatcouldn't be modeled in Kgp that falls within the second interactionsurface. FIG. 7 shows the Kgp model with the thicker ribbons showingsurface accessible sequences around the proteinase active site of Kgp,the surface accessible sequences were found to be Asp388-Gln394,Leu421-Ala423, Ala443-Glu447 with Ala451, Asn510-Trp513, andIle570-Gly577 with Tyr580. From the model (FIG. 6 ) it is evident thatalong with KAS2 (A) three other sequences KAS4 (Asp388-Va1395) (B), KAS5(Asn510-Asp516) (C) and KAS6 (1Ie570-Tyr580) (D) are prominent and ofsufficient length to be vaccine targets. Thus a recombinant chimeraprotein can be produced that has each of these peptides in sequence andjoined on to the N-terminus of KLA1 to produce multiKAS-KLA1, that canbe used to induce an immune response and hence to protect against P.gingivalis related diseases or conditions.

Example 10

Process for Modelling Arg-X-Proteinase to Identify Immunogenic RegionsFlanking the Catalytic Site.

The Arg-X proteinase three dimensional structure was determinedaccording to the methods of Eichinger A, Beisel H G, Jacob U, Huber R,Medrano F J, Banbula A, Potempa J, Travis J, Bode W. Crystal structureof gingipain R: an Arg-specific bacterial cysteine proteinasewith acaspase-like fold. EMBO J. 1999 Oct. 15; 18(20):5453-62

Example 11

The following is an example of a toothpaste formulation containingantibodies.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Glycerol 20.0 Sodiumcarboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroylsarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Chlorhexidine gluconate0.01 Dextranase 0.01 Goat serum containing specific antibodies 0.2 Waterbalance

Example 12

The following is an example of a toothpaste formulation.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase0.01 Bovine serum containing specific antibodies 0.2 Water balance

Example 13

The following is an example of a toothpaste formulation.

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide1.0 Sucrose monolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01Bovine milk Ig containing specific antibodies 0.1 Water balance

Example 14

The following is an example of a toothpaste formulation.

Ingredient % w/w Sorbitol 22.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0Gantrez 19.0 Water (deionised) 2.69 Sodium Monofluorophosphate 0.76Sodium saccharine 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavouroil 0.95 Mouse monoclonal antibodies 0.3 sodium lauryl sulphate 2.00

Example 15

The following is an example of a liquid toothpaste formulation.

Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0Flavour 1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3Chlorhexidine gluconate 0.01 Ethanol 3.0 Equine Ig containing specificantibodies 0.2 Linolic acid 0.05 Water balance

Example 16

The following is an example of a mouthwash formulation.

Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.3 Rabbit Ig containing specific antibodies 0.2 Waterbalance

Example 17

The following is an example of a mouthwash formulation.

Ingredient % w/w Gantrez S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodiummonofluorophosphate 0.05 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.2 Mouse monoclonal antibodies 0.3 Water balance

Example 18

The following is an example of a lozenge formulation.

Ingredient % w/w Sugar 75-80 Corn syrup  1-20 Flavour oil 1-2 NaF0.01-0.05 Mouse monoclonal antibodies 0.3 Mg stearate 1-5 Water balance

Example 19

The following is an example of a gingival massage cream formulation.

Ingredient % w/w White petrolatum 8.0 Propylene glycol 4.0 Stearylalcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0Sucrose monostearate 0.5 Chlorohexidine gluconate 0.1 Mouse monoclonalantibodies 0.3 Water balance

Example 20

The following is an example of a chewing gum formulation.

Ingredient % w/w Gum base 30.0 Calcium carbonate 2.0 Crystallinesorbitol 53.0 Glycerine 0.5 Flavour oil 0.1 Mouse monoclonal antibodies0.3 Water balance

Example 21

The following is an example of a pharmaceutical formulation

Ingredient % w/w Humanised specific monoclonal antibodies 10 Sterilephosphate buffered saline 90

Example 22

The following is an example of a periodontal gel formulation.

Ingredient % w/w Pluronic F127 20.0 Stearyl alcohol 8.0 Specificantibodies 3.0 Colloidal silicon dioxide (Aerosil 200) 1.0 Chlorhexidinegluconate 0.1 Water balance

It should be understood that while the invention has been described indetails herein, the examples are for illustrative purposes only. Othermodifications of the embodiments of the present invention that areobvious to those skilled in the art of molecular biology, dentaldiagnostics, and related disciplines are intended to be within the scopeof the invention.

REFERENCES

-   1. McKee, A. S., A. S. McDermid, A. Baskerville, A. B.    Dowsett, D. C. Ellwood, and P. D. Marsh. 1986. Effect of hemin on    the physiology and virulence of Bacteroides gingivalis W50. Infect.    Immun. 52:349-355.-   2. Slots, J. 1982. Importance of black-pigmented Bacteroides in    human periodontal disease. Host parasite interactions in periodontal    diseases. American Society for Microbiology.-   3. O'Brien-Simpson, N. M., R. Pathirana, R. A. Paolini, Y.-Y.    Chen, P. D. Veith, T. V., R. N. Pike, N. Alley, and E. C.    Reynolds. 2005. An immune response directed to proteinase and    adhesin functional epitopes protects against Porphyromonas    gingivalis-induced bone loss. Journal of Immunology 175:3980-3989.-   4. Baker, P. J., R. T. Evans, and D. C. Roopenian. 1994. Oral    infection with Porphyromonas gingivalis and induced alveolar bone    loss in immunocompetent and severe combined immunodeficient mice.    Arch Oral Biol 39:1035-1040.

1-70. (canceled)
 71. A chimeric or fusion protein for inducing an immuneresponse to P. gingivalis, the protein comprising a first peptide joineddirectly or through a linker to a second peptide or polypeptide,wherein: (A) said first peptide comprises a region of a P. gingivalistrypsin-like enzyme, wherein the first peptide comprises part of, or allof a sequence that is the same as, or at least 90% homologous to thesequence shown in any one of SEQ ID Nos: 1, 2, or 27 to 34; and (B) saidsecond peptide or polypeptide comprises a P. gingivalis adhesin domainsequence or fragment thereof, wherein the second peptide or polypeptidecomprises part of, or all of a sequence that is the same as, or at least90% homologous to the sequence shown in any one of SEQ ID Nos: 35 to 39.72. The chimeric or fusion protein according to claim 71, wherein saidfirst peptide comprises a sequence selected from the group consisting ofSEQ ID NOs: 27 to
 34. 73. The chimeric or fusion protein according toclaim 71, wherein said second peptide or polypeptide comprises asequence selected from the group consisting of SEQ ID NOs: 35 to
 37. 74.The chimeric or fusion protein according to claim 71, wherein: (a) saidfirst peptide comprises a sequence that is the same as or at least 90%homologous to a sequence selected from the group consisting of SEQ IDNOs: 27-30 and said second peptide or polypeptide comprises a sequencethat is the same as or 90% homologous to a sequence selected from thegroup consisting of SEQ ID NOs: 36-37; or (b) said first peptidecomprises a sequence that is the same as or at least 90% homologous to asequence selected from the group consisting of SEQ ID NOs: 31-34 andsaid second peptide or polypeptide comprises a sequence that is the sameas or at least 90% homologous to SEQ ID NO:39.
 75. The chimeric orfusion protein according to claim 71, wherein: (a) said first peptidecomprises a sequence that is the same or at least 90% homologous to thesequence shown in SEQ ID NO: 28 and said second peptide or polypeptidecomprises a sequence that is the same or at least 90% homologous to thesequence shown in SEQ ID NO: 37; or (b) said first peptide comprises asequence that is the same or at least 90% homologous to the sequenceshown in SEQ ID NO: 27 and said second peptide or polypeptide comprisesa sequence that is the same or at least 90% homologous to the sequenceshown in SEQ ID NO:
 36. 76. A chimeric or fusion protein according toclaim 71, wherein: the C-terminal residue of said first peptide iscovalently linked directly or through a linker that is either (i) up to15 amino acids in length, or (ii) less than 5 amino acids in length, toeither (a) the N-terminal residue or (b) the C-terminal residue of saidsecond peptide or polypeptide.
 77. A chimeric or fusion proteinaccording to claim 71, wherein: the N-terminal residue of said firstpeptide is covalently linked directly or through a linker that is either(i) up to 15 amino acids in length, or (ii) less than 5 amino acids inlength, to either (a) the N-terminal residue or (b) the C-terminalresidue of said second peptide or polypeptide.
 78. A compositioncomprising a chimeric or fusion protein according to claim
 71. 79. Thecomposition according to claim 78, further comprising an adjuvant.
 80. Amethod of preventing or reducing the incidence or severity of a P.gingivalis-related condition or disease in a subject, comprisingadministering to the subject a chimeric or fusion protein of claim 71.81. An antibody raised against the chimeric or fusion protein of claim71.
 82. The antibody according to claim 81, wherein the antibody is amonoclonal antibody.
 83. A method of preventing or reducing the severityof a P. gingivalis-related disease or condition in a subject, comprisingadministering to the subject the antibody of claim
 71. 84. A nucleicacid molecule comprising a sequence encoding the chimeric or fusionprotein of claim
 71. 85. A method for the diagnosis or monitoring of aP. gingivalis-related condition or disease in a subject, which comprisesassaying a biological sample from said subject with the chimeric orfusion protein of claim 71 to detect anti-P. gingivalis antibodies. 86.A method for the diagnosis or monitoring of a P. gingivalis-relatedcondition or disease in a subject, which comprises assaying a biologicalsample from said subject with the antibody according to claim 81, todetect the presence of P. gingivalis.